U.S. patent application number 11/728494 was filed with the patent office on 2008-07-03 for compositions and methods for regulating abscisic acid-induced closure of plant stomata.
This patent application is currently assigned to Penn State Research Foundation. Invention is credited to Sarah M. Assmann, Jiaxu Li.
Application Number | 20080163391 11/728494 |
Document ID | / |
Family ID | 37991412 |
Filed Date | 2008-07-03 |
United States Patent
Application |
20080163391 |
Kind Code |
A1 |
Assmann; Sarah M. ; et
al. |
July 3, 2008 |
Compositions and methods for regulating abscisic acid-induced
closure of plant stomata
Abstract
A novel gene, AAPK, is disclosed. Loss of function of the
protein encoded by AAPK is associated with reduced sensitivity to
abscisic acid-induced stomatal closure in plants. Also disclosed
are transgenic plants and mutants having altered sensitivity to
abscisic acid-mediated transpiration and other desirable agronomic
features. The regulation of transpiration provided by the present
invention is different from that of previously described mechanisms
to control transpiration in plants.
Inventors: |
Assmann; Sarah M.; (State
College, PA) ; Li; Jiaxu; (State College,
PA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR, 2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
Penn State Research
Foundation
|
Family ID: |
37991412 |
Appl. No.: |
11/728494 |
Filed: |
March 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09606736 |
Jun 29, 2000 |
7211436 |
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11728494 |
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60142039 |
Jul 1, 1999 |
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60176245 |
Jan 14, 2000 |
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60192499 |
Mar 28, 2000 |
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Current U.S.
Class: |
800/278 ;
800/298 |
Current CPC
Class: |
C12Q 1/6895 20130101;
C12N 9/1205 20130101; C12N 15/8293 20130101 |
Class at
Publication: |
800/278 ;
800/298 |
International
Class: |
C12N 15/87 20060101
C12N015/87; A01H 5/00 20060101 A01H005/00 |
Goverment Interests
[0002] Pursuant to 35 U.S.C. .sctn.202(c), it is acknowledged that
the U.S. Government has certain rights in the invention described
herein, which was made in part with funds from the National Science
Foundation, Grant Nos. MCB-9316319 and MCB-9874438.
Claims
1-25. (canceled)
26. A genetically altered plant possessing increased sensitivity to
ABA-induced stomatal closure as compared with an equivalent but
unaltered plant, comprising an AAPK that is increased in amount or
activity as compared with the unaltered plant.
27. The genetically altered plant of claim 26, produced by
subjecting a population of plants to mutagenesis and selecting a
mutagenized plant wherein the AAPK is largely nonfunctional or
absent.
28. The genetically altered plant of claim 26, produced by
transforming cells of the plant with a transgene that causes the
plant's endogenous AAPK to become largely nonfunctional or absent,
and regenerating the plant from the transformed cells.
29. The genetically altered plant of claim 28, wherein expression
of the transgene is inducible.
30-37. (canceled)
38. A method to decrease transpiration in a plant comprising
increasing amount or activity of an AAPK in guard cells of the
plant, thereby increasing sensitivity of the plant to ABA-induced
stomatal closure, resulting in the decreased transpiration.
39. The method of claim 38, wherein the AAPK amount or activity is
increased by the addition of at least one transgene to the plant
genome.
40. A fertile plant produced by the method of claim 38.
Description
[0001] This application claims priority to U.S. Provisional
Application Nos. 60/142,039, filed Jul. 1, 1999, 60/176,245, filed
Jan. 14, 2000 and 60/192,499, filed Mar. 28, 2000, the entireties
of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of molecular
biology of plants. More specifically, it relates to the regulation
of gas exchange and transpirational water loss in plants possessing
stomata.
BACKGROUND OF THE INVENTION
[0004] Various scientific and scholarly articles are referred to
throughout the specification. These articles are incorporated by
reference herein to describe the state of the art to which this
invention pertains.
[0005] In terrestrial plants, water is transported, from the roots
to the leaves, down a water potential gradient from the soil to the
air. Transpiration, or loss of water from the leaves, helps create
lowered osmotic potential in the leaves, effectively drawing water
from the xylem to the mesophyll cells into the air spaces in the
leaves. Estimates are that 90% or more of the water taken up by
plants is lost to the air via transpiration.
[0006] Transpirational loss of water by evaporation occurs mainly
through the pores, called stomata, primarily located in the lower
epidermis of the leaves. Each stoma is surrounded by two guard
cells, which control the opening and closure of the stomata by
their relative turgor pressure. The cell wall properties of guard
cells allow them to deform such that when the guard cells develop
turgor pressure, the stoma is opened, but when the guard cells lose
turgor, the stoma closes.
[0007] The rate of evaporation of water from the air spaces of the
leaf to the outside air depends on the water potential gradient
between the leaf and the outside air. Environmental factors which
directly influence the aperture of the plant's stomata affect its
transpiration rate. Such factors include light conditions, relative
humidity of the air, temperature, water status of the plant,
CO.sub.2 concentration, relative concentration of certain ions, and
concentration of abscisic acid (ABA).
[0008] Abscisic acid is a multifunctional phytohormone involved in
a variety of important protective functions including bud dormancy,
seed dormancy and/or maturation, abscission of leaves and fruits,
and response to a wide variety of biological stressors (e.g. cold,
heat, salinity, and drought). It is also responsible for regulating
stomatal closure by a mechanism independent of CO.sub.2
concentration.
[0009] ABA is synthesized rapidly in response to water stress in
plants, and is stored in the guard cells. During drought, ABA
alteration of guard cell ion transport promotes stomatal closure
and also prevents stomatal opening, thus reducing transpirational
water loss. At the biochemical level, it is believed that the
hormone sets off a variety of biological messages that require or
include a protein phopsphorylation cascade. One member of this
cascade was identified in guard cells of Vicia faba as an
ABA-activated, calcium-independent protein kinase. (Li &
Assmann, Plant Cell 8: 2359-2368, 1996; Mori & Muto, Plant
Physiol. 113: 833-839, 1997). The kinase was identified by SDS
polyacrylamide gel electrophoresis as a 48 kDa protein, but was not
further isolated or characterized. It exhibited ABA-activated
autophosphorylation and kinase activity.
[0010] Stomata simultaneously regulate both the transpiration of
water and the exchange of gases for photosynthesis. Open stomata
allow for maximum gas exchange rate so that photosynthetic
reactions may proceed more quickly, however under these conditions,
water loss will be maximal. On the other hand, closed stomata
minimize transpirational water loss but also substantially reduce
photosynthetic reaction rates. Paradoxically, the plant undergoes a
continual trade-off between maximizing CO.sub.2 uptake for carbon
fixation, and minimizing desiccating water loss. Thus, the ability
to control stomatal opening and closure could be of tremendous
agronomic significance.
[0011] Several studies in the literature provide examples of the
benefits of selecting for increased stomatal conductance under
certain conditions. One system that has been studied extensively
comprises eight lines of Pima cotton (Gossypium barbadense)
obtained over 40 years of selection and showing a 3-fold range in
yield. These and additional studies have confirmed the association
of higher conductance with higher yield, and its genetic basis, in
both Pima and Upland (Gossypium hirsutum) cotton. A similar
correlation of increased yield, increased stomatal conductance, and
decreased canopy temperatures has also been observed in a
historical series of bread wheat cultivars. Taken collectively,
this body or research suggests that selection or genetic
engineering of plants to achieve increased stomatal conductance may
be of widespread utility for crop plants grown under irrigation
under supra-optimal temperatures.
[0012] The plant hormone abscisic acid (ABA) causes stomatal
closure during periods of reduced water availability by reducing
the ion and water content of the pair of guard cells that flanks
each stoma. However, even when plants are well-watered, ABA still
limits stomatal aperture, as shown by the fact that mutants of
tomato and Arabidopsis that are deficient in either ABA-production
or ABA-sensing have larger stomatal apertures than wild-type
plants, even when water is plentiful. In other words, the ABA
response is protective; always somewhat limiting to water loss, but
thus unavoidably, also limiting to CO.sub.2 uptake. This
ABA-mediated limitation of water loss is of no benefit however, to
the grower who irrigates crops so that they are always
well-watered. For those crops, such as many of the agricultural
crops that are grown in arid or semi-arid regions, if this
endogenous ABA-response of the stomata were "turned off", crop
yield could be increased, or the time for the plant to reach
maturity decreased by removing the limits on increased CO.sub.2
uptake and fixation.
[0013] Many crops, for example feed corn and wheat, are dried in
the field before harvest. Other crops, such as tobacco and dried
fruits such as raisins and prunes, are dried immediately
post-harvest. It would be advantageous to growers to be able to
accelerate or control the rate of crop drying.
[0014] For example, at the end of the growing season, it might be
advantageous to dry the plants as quickly as possible, to minimize
exposure to adverse weather conditions. However, water stress
inevitably triggers ABA production/redistribution in the plant,
leading to stomatal closure, which slows the rate of water loss,
thus slowing the rate of crop drying. Therefore, it would be
advantageous to growers if this ABA-triggered stomatal closure
response could be prevented or controlled.
[0015] In other cases, post-harvest, for many fruits, vegetables,
and for cut-flowers, it is advantageous for the produce to dry out
as slowly as possible, to retain freshness during transport,
distribution, and purchase of the product. In these situations, it
would be advantageous if the ABA-induced stomatal closure response
could be enhanced. This could significantly extend the shelf life
of the product.
[0016] The theoretical solution to the problems posed above is for
growers to be able to precisely control the plant's transpiration
via ABA-responsiveness of the guard cells/stomata. Ideally this
control should be: [0017] 1. specific to the Guard Cells. It should
not disrupt the many other effects of ABA on plant growth and
development. [0018] 2. specific to ABA. There are many other
stimuli that guard cells respond to in the control of stomatal
aperture, for example, light and decreased intracellular
concentrations of CO.sub.2 drive stomatal opening, and conversely,
darkness and high CO.sub.2 concentrations drive stomatal closure.
For crops under irrigation for example, the grower would still want
the stomata to close in response to darkness, because in darkness
there is no photosynthesis anyway, and open stomata during the
night would simply waste irrigation water and thus money. [0019] 3.
Inducible, Titratable, and Reversible. To be of greatest utility,
the grower would want to be able to control "when" and "how much"
the guard cells respond to ABA. Ideally, the grower would be able
to open and close the stomata depending on the prevailing
environmental conditions and desired results for his crop.
[0020] In present practice, growers have only limited control of
rates of water loss from plants, mainly by controlling irrigation
regimens in the field, and controlling environmental conditions
during shipping, handling and storage of the product. By
capitalizing on the present invention, growers could choose when
plants would retain their maximum hydrated status (e.g. during
times of water restriction, or for shipping of fresh produce), and
when plants could be induced to dry out more quickly (e.g. as
required for crops that are dried in the field before harvest).
[0021] Mutants have been identified in Arabidopsis, which display
reduced rates of ABA production (aba mutants; Koornneefet al. 1982,
Theor. Appl. Genet. 61:385-393) or ABA sensing (abi mutants;
Koornneefet al. 1984 Physiol. Plant. 61:377-383). While these
plants exhibit increased rates of water loss, the mutations are
pleiotropic and this is a disadvantage. For example, the aba and
abi mutants have reduced seed dormancy, and so the viability of the
seed is likely to be reduced, a severe problem limiting any
commercial application.
[0022] The isolation of novel mutants and genes that encode altered
ABA-mediation of transpirational water loss will broaden the range
of options for growers. It would be particularly advantageous to
isolate mutants or genes involved in altered ABA-mediation of
transpiration without spontaneously occurring abnormal responses to
other roles of ABA or abnormal responses to factors such as light
levels and concentrations of CO.sub.2. Novel regulatory mutants are
likely to have distinct induction of unique subsets of genes. The
isolation of mutants will yield the critical gene(s) involved with
altered ABA-mediation of transpiration, which can be used to
transgenically transfer the novel trait to other species.
SUMMARY OF THE INVENTION
[0023] Provided in the present invention is a novel nucleic acid
molecule (referred herein as AAPK), which is associated with
regulation of transpiration by the hormone abscisic acid (ABA) in
plants. The invention further provides transgenic plants and
mutants having modified ABA-mediated stomatal closure. In these
plants, ABA-mediated stomatal closure is modified in a manner that
is independent of CO.sub.2- and light-mediated responses of
transpiration, as measured by changes in stomatal aperture.
[0024] According to one aspect of the present invention, a nucleic
acid molecule encoding an ABA-activated protein kinase, AAPK, is
provided. An -exemplary AAPK-encoding nucleic acid molecule of the
invention is that of Vicia faba, a food crop of major importance in
the Middle East. Also exemplified are homologs of the gene in
Arabidopsis thaliana. The invention farther provides homologs of
the exemplified AAPK, having a level of nucleotide sequence or
amino acid sequence identity with the exemplified AAPK nucleic
acids or encoded AAPK proteins, specifically at certain regions of
the coding sequence, that clearly distinguish the homologs as AAPK
homologs, as opposed to other kinases. Preferably, these homologs
comprise nucleotide or amino acid sequences at least 60%,
preferably 67%, more preferably 70% and even more preferably 80%
identical to the Vicia faba and Arabidopsis AAPK nucleic acid and
AAPK amino acid sequences set forth herein.
[0025] Also provided in accordance with the present invention is a
disrupted gene product of the AAPK gene. In a preferred embodiment,
the disrupted gene product comprises lost or reduced activity of
the AAPK protein. Reduction in amount or activity of AAPK in plants
results in decreased sensitivity of the plants to ABA-induced
stomatal closure, but does not affect the plants's sensitivity to
dark- or CO.sub.2-induced stomatal closure.
[0026] According to another aspect of the invention, an
oligonucleotide molecule of at least 15 nucleotides in length,
preferably at least 20 nucleotides in length, and most preferably
at least 30 nucleotides in length, that is identical in sequence to
a portion of an AAPK nucleic acid, is provided. In a preferred
embodiment, the invention provides a nucleic acid molecule of at
least 15, preferably 20, and most preferably 30 or more nucleotides
in length, that is identical to or complementary to a consecutive
15, 20 or 30 nucleotide portion, respectively, of the sequence set
out in one of SEQ ID NOS:1, 3 or 6.
[0027] According to other aspects of the invention, an isolated
polypeptide produced by expression of a nucleic acid molecule of
the invention is provided. Also featured are antibodies
immunologically specific for such a polypeptide.
[0028] According to another aspect of the invention, a vector for
transforming a plant cell, comprising a nucleic acid molecule of
the invention, is provided. Also featured are plant cells
transformed with the vector, and intact fertile plants regenerated
from the plant cells. It will be appreciated by persons of skill in
the art, that various portions of such genetically altered plants
are also encompassed by the present invention. These include, but
are not limited to, roots, modified roots (e.g., tubers), stems,
leaves, flowers, fruits and seeds, and components thereof, e.g.,
extracts or oils.
[0029] According to another aspect of the invention, a genetically
altered plant is provided, which possesses decreased sensitivity to
ABA-induced stomatal closure as compared with an equivalent but
unaltered plant. These genetically altered plants contain an AAPK
that is largely nonfunctional or absent. In one embodiment, the
plant is produced by subjecting a population of plants to
mutagenesis and selecting a mutagenized plant wherein the AAPK is
largely nonfunctional or absent. In a preferred embodiment, the
plant is produced by transforming cells of the plant with a
transgene that causes the plant's endogenous AAPK to become largely
nonfunctional or absent, and regenerating the plant from the
transformed cell. In a particularly preferred embodiment,
expression of the transgene is inducible.
[0030] According to another aspect of the invention, a genetically
altered plant possessing increased sensitivity to ABA-induced
stomatal closure as compared with an equivalent but unaltered
plant, is provided. Plants of this type contain an AAPK that is
increased in amount or activity as compared with the unaltered
plant. In one embodiment, these plants are produced by subjecting a
population of plants to mutagenesis and selecting a mutagenized
plant wherein the AAPK is largely nonfunctional or absent. In a
preferred embodiment, the plants are produced by transforming cells
of the plant with a transgene that causes the plant's endogenous
AAPK to become largely nonfunctional or absent, and regenerating
the plant from the transformed cells. In a particularly preferred
embodiment, expression of the transgene is inducible.
[0031] Another aspect of the invention features a method to
increase transpiration in a plant. The method comprises reducing or
preventing function of an AAPK in guard cells of the plant, thereby
reducing sensitivity of the plant to ABA-induced stomatal closure,
resulting in the increased transpiration. Conversely, a method is
provided to decrease transpiration in a plant, comprising
increasing function of AAPK in guard cells of the plant, thereby
increasing sensitivity of the plant to ABA-induced stomatal
closure, resulting in decreased transpiration.
[0032] Other features and advantages of the present invention will
be better understood by reference to the drawings, detailed
description and examples that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1. Alignment of the deduced AAPK amino acid sequence
with those of homologous protein kinases. GenBank accession numbers
for the nucleic acid molecules encoding the displayed amino acid
sequences are: AAPK (AF186020), Arabidopsis Atpk (L05562, S71172),
tobacco WAPK (AF032465), soybean SPK-4 (L38855), rice REK
(AB002109), ice plant MK9 (Z26846), and wheat PKABA1 (M94726).
Sequence ID Numbers for the displayed sequences are as follows:
AAPK is SEQ ID NO:2 (encoded by SEQ ID NO:1); Atpk is SEQ ID NO:4
(encoded by SEQ ID NO:3); WAPK is SEQ ID NO: 11; SPK-4 is SEQ ID
NO:12; REK is SEQ ID NO:13; MK9 is SEQ ID NO:14; PKABA1 is SEQ ID
NO: 10 (encoded by SEQ ID NO:9). Amino acids are highlighted when
there are at least four identical residues among the seven
sequences. Conserved subdomains of the protein kinase family are
indicated by roman numerals. Peptide sequences obtained by tandem
mass spectrometry are marked by lines. Peptide regions used for
designing degenerate PCR primers are indicated by arrows. Sequences
were aligned by the Clustal method in MegAlign (DNASTAR, Madison,
Wis.). Numbers indicate amino acid positions.
[0034] FIG. 2. Alignment of the deduced amino acid sequence of AAPK
from Vicia faba (SEQ ID NO:2) with the amino acid sequence of a
homologous protein kinase from Arabidopsis thaliana (SEQ ID NO:5).
Query sequence=AAPK (GI 6739629), Length=349 amino acids. Subject
sequence=A. thaliana protein kinase, Length=357 amino acids
[GenBank Accession Number CAA19877 (GI 3297819)]. Comparison was
done using the Blast 2 alignment program at NCBI, with following
default parameters: Matrix: BLOSUTM62, Gap Penalties: Existence:
11, Extension: 1. Identities=270/348 (77%), Positives=311/348
(88%), Gaps=1/348 (0%). The sequence shown between the Query and
the Subject sequences shows the consensus sequence. A letter
indicates identity, a `+` indicates a similarity, while a blank
space indicates the two sequences are different at that
residue.
[0035] FIG. 3. Alignment of the deduced amino acid sequence of AAPK
from Vicia faba (SEQ ID NO:2) with the deduced amino acid sequence
from a gene encoding a homologous protein kinase from Arabidopsis
thaliana (SEQ ID NO:7). Query sequence=AAPK (GI 6739629),
Length=349 amino acids. Subject sequence=deduced amino acid
sequence from A. thaliana L05561 clone, Length=362 amino acids
[GenBank Accession Number L05561 (GI 166817)]. Comparison was done
using the Blast 2 alignment program at NCBI, with following default
parameters: Matrix: BLOSUM62, Gap Penalties: Existence: 11,
Extension: 1. Identities=273/353 (77%), Positives=318/353 (89%),
Gaps=4/353 (1%). The sequence shown between the Query and the
Subject sequences shows the consensus sequence. A letter indicates
identity, a `+` indicates a similarity, while a blank space
indicates the two sequences are different at that residue.
[0036] FIG. 4. Alignment of the deduced amino acid sequence of AAPK
from Vicia faba (SEQ ID NO:2) with the amino acid sequence of a
homolog, Protein Kinase SPK-2, from Arabidopsis thaliana (SEQ ID
NO:8). Query sequence=AAPK (GI 6739629), Length=349 amino acids.
Subject sequence=amino acid sequence from A. thaliana, Length=362
amino acids [GenBank Accession Number S56718 (GI 1362002)].
Comparison was done using the Blast 2 alignment program at NCBI,
with following default parameters: Matrix: BLOSUM62, Gap Penalties:
Existence: 11, Extension: 1. Identities=273/353 (77%),
Positives=318/353 (89%), Gaps=4/353 (1%). The sequence shown
between the Query and the Subject sequences shows the consensus
sequence. A letter indicates identity, a `+` indicates a
similarity, while a blank space indicates the two sequences are
different at that residue.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0037] Various terms relating to the biological molecules of the
present invention are used hereinabove and also throughout the
specifications and claims.
[0038] With respect to the genotypes of the invention, the term
"AAPK" is used to designate the naturally-occurring or wild-type
genotype. This genotype has the phenotype of naturally-occurring
sensitivity to the effects of ABA. Where used hereinabove and
throughout the specifications and claims, the term "AAPK" refers to
the protein product of the AAPK gene.
[0039] In reference to the mutants of the invention, the term "null
mutant" or "loss-of-function mutant" is used to designate an
organism or genomic DNA sequence with a mutation that causes the
product of the gene of interest to be non-functional or largely
absent. Such mutations may occur in the coding and/or regulatory
regions of the gene of interest, and may be changes of individual
residues, or insertions or deletions of regions of nucleic acids.
These mutations may also occur in the coding and/or regulatory
regions of other genes which may regulate or control the gene of
interest and/or its encoded gene product so as to cause said gene
product to be non-functional or largely absent.
[0040] With reference to certain of the DNA constructs of the
invention, the terms "pGFP", "pAAPK-GFP" and "pAAPK(K43A)-GFP"
refer to constructs made from the green fluorescent protein (GFP)
expression vector, pGFP, which allows cells transformed with the
pGFP to express the readily-detected green fluorescent protein.
Where used herein, "pAAPK-GFP" refers to a pGFP expression vector
with the sequence encoding AAPK inserted upstream of and in-frame
with the sequence encoding the GFP, such that both proteins can be
expressed in cells transformed with this construct. Where used
herein, "pAAPK(K43A)-GFP" refers to a pGFP expression vector with
the sequence encoding an AAPK, modified such that the lysine
residue at position 43 in the ATP binding site is changed to an
alanine, inserted upstream of and in-frame with the sequence
encoding the GFP, such that both proteins can be expressed in cells
transformed with this construct.
[0041] With reference to nucleic acids of the invention, the term
"isolated nucleic acid" is sometimes used. This term, when applied
to genomic DNA, refers to a DNA molecule that is separated from
sequences with which it is immediately contiguous (in the 5' and 3'
directions) in the naturally-occurring genome of the organism from
which it was derived. For example, the "isolated nucleic acid" may
comprise a DNA molecule inserted into a vector, such as a plasmid
or virus vector, or integrated into the genomic DNA of a procaryote
or eukaryote. An "isolated nucleic acid molecule" may also comprise
a cDNA molecule or a synthetic DNA molecule.
[0042] With respect to RNA molecules of the invention, the term
"isolated nucleic acid" primarily refers to an RNA molecule encoded
by an isolated DNA molecule as defined above. Alternatively, the
term may refer to an RNA molecule that has been sufficiently
separated from RNA molecules with which it would be associated in
its natural state (i.e., in cells or tissues), such that it exists
in a "substantially pure" form.
[0043] Nucleic acid sequences and amino acid sequences can be
compared using computer programs that align the similar sequences
of the nucleic or amino acids thus define the differences. In
preferred methodologies, the BLAST programs (NCBI) and parameters
used therein are employed to align nucleotide and amino acid
sequences. However, equivalent alignments and similarity/identity
assessments can be obtained through the use of any standard
alignment software. For instance, the DNAstar system (Madison,
Wis.) may be used to align sequence fragments of genomic or other
DNA sequences. Alternatively, GCG Wisconsin Package version 9.1,
available from the Genetics Computer Group in Madison, Wis. and the
default parameters used (gap creation penalty=12, gap extension
penalty=4) by that program may also be used to compare sequence
identity and similarity.
[0044] The term "substantially the same" refers to nucleic acid or
amino acid sequences having sequence variation that do not
materially affect the nature of the protein (i.e. the structure,
stability characteristics and/or biological activity of the
protein). With particular reference to nucleic acid sequences, the
term "substantially the same" is intended to refer to the coding
region and to conserved sequences governing expression, and refers
primarily to degenerate codons encoding the same amino acid, or
alternate codons encoding conservative substitute amino acids in
the encoded polypeptide. With reference to amino acid sequences,
the term "substantially the same" refers generally to conservative
substitutions and/or variations in regions of the polypeptide not
involved in determination of structure or function.
[0045] The terms "percent identical" and "percent similar" are also
used herein in comparisons among amino acid and nucleic acid
sequences. When referring to amino acid sequences, "percent
identical" refers to the percent of the amino acids of the subject
amino acid sequence that have been matched to identical amino acids
in the compared amino acid sequence by a sequence analysis program.
"Percent similar" refers to the percent of the amino acids of the
subject amino acid sequence that have been matched to identical or
conserved amino acids. Conserved amino acids are those which differ
in structure but are similar in physical properties such that the
exchange of one for another would not appreciably change the
tertiary structure of the resulting protein. Conservative
substitutions are defined by Taylor (1986, J. Theor. Biol.
119:205). When referring to nucleic acid molecules, "percent
identical" refers to the percent of the nucleotides of the subject
nucleic acid sequence that have been matched to identical
nucleotides by a sequence analysis program.
[0046] With respect to protein, the term "isolated protein" or
"isolated and purified protein" is sometimes used herein. This term
refers primarily to a protein produced by expression of an isolated
nucleic acid molecule of the invention. Alternatively, this term
may refer to a protein which has been sufficiently separated from
other proteins with which it would naturally be associated, so as
to exist in "substantially pure" form. In this regard, "isolated"
or "isolated and purified" also refers to its separation or removal
from a chromatography column matrix or a gel, such as a
polyacrylamide gel. That is, a polypeptide that has been separated
by chromatography or polyacrylamide gel electrophoresis, but is not
eluted from the matrix or gel, is not considered "isolated" or
"isolated and purified".
[0047] With respect to antibodies of the invention, the terms
"immunologically specific" or "specific" refer to antibodies that
bind to one or more epitopes of a protein of interest, but which do
not substantially recognize and bind other molecules in a sample
containing a mixed population of antigenic biological
molecules.
[0048] With respect to single-stranded nucleic acid molecules, the
term "specifically hybridizing" refers to the association between
two single-stranded nucleic acid molecules of sufficiently
complementary sequence to permit such hybridization under
pre-determined conditions generally used in the art (sometimes
termed "substantially complementary"). In particular, the term
refers to hybridization of an oligonucleotide with a substantially
complementary sequence contained within a single-stranded DNA or
RNA molecule, to the substantial exclusion of hybridization of the
oligonucleotide with single-stranded nucleic acids of
non-complementary sequence.
[0049] A "coding sequence" or "coding region" refers to a nucleic
acid molecule having sequence information necessary to produce a
gene product, when the sequence is expressed.
[0050] The term "operably linked" or "operably inserted" means that
the regulatory sequences necessary for expression of the coding
sequence are placed in a nucleic acid molecule in the appropriate
positions relative to the coding sequence so as to enable
expression of the coding sequence. This same definition is
sometimes applied to the arrangement of other transcription control
elements (e.g. enhancers) in an expression vector.
[0051] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0052] The terms "promoter", "promoter region" or "promoter
sequence" refer generally to transcriptional regulatory regions of
a gene, which may be found at the 5' or 3' side of the coding
region, or within the coding region, or within introns. Typically,
a promoter is a DNA regulatory region capable of binding RNA
polymerase in a cell and initiating transcription of a downstream
(3' direction) coding sequence. The typical 5' promoter sequence is
bounded at its 3' terminus by the transcription initiation site and
extends upstream (5' direction) to include the minimum number of
bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence is a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
[0053] A "vector" is a replicon, such as plasmid, phage, cosmid, or
virus to which another nucleic acid segment may be operably
inserted so as to bring about the replication or expression of the
segment.
[0054] The term "nucleic acid construct" or "DNA construct" is
sometimes used to refer to a coding sequence or sequences operably
linked to appropriate regulatory sequences and inserted into a
vector for transforming a cell. This term may be used
interchangeably with the term "transforming DNA". Such a nucleic
acid construct may contain a coding sequence for a gene product of
interest, along with a selectable marker gene and/or a reporter
gene.
[0055] The term "selectable marker gene" refers to a gene encoding
a product that, when expressed, confers a selectable phenotype such
as antibiotic resistance on a transformed cell.
[0056] The term "reporter gene" refers to a gene that encodes a
product which is easily detectable by standard methods, either
directly or indirectly.
[0057] A "heterologous" region of a nucleic acid construct is an
identifiable segment (or segments) of the nucleic acid molecule
within a larger molecule that is not found in association with the
larger molecule in nature. Thus, when the heterologous region
encodes a mammalian gene, the gene will usually be flanked by DNA
that does not flank the mammalian genomic DNA in the genome of the
source organism. In another example, a heterologous region is a
construct where the coding sequence itself is not found in nature
(e.g., a cDNA where the genomic coding sequence contains introns,
or synthetic sequences having codons different than the native
gene). Allelic variations or naturally-occurring mutational events
do not give rise to a heterologous region of DNA as defined herein.
The term "DNA construct", as defined above, is also used to refer
to a heterologous region, particularly one constructed for use in
transformation of a cell.
[0058] A cell has been "transformed" or "transfected" by exogenous
or heterologous DNA when such DNA has been introduced inside the
cell. The transforming DNA may or may not be integrated (covalently
linked) into the genome of the cell. In prokaryotes, yeast, and
mammalian cells for example, the transforming DNA may be maintained
on an episomal element such as a plasmid. With respect to
eukaryotic cells, a stably transformed cell is one in which the
transforming DNA has become integrated into a chromosome so that it
is inherited by daughter cells through chromosome replication. This
stability is demonstrated by the ability of the eukaryotic cell to
establish cell lines or clones comprised of a population of
daughter cells containing the transforming DNA. A "clone" is a
population of cells derived from a single cell or common ancestor
by mitosis. A "cell line" is a clone of a primary cell that is
capable of stable growth in vitro for many generations.
II. Description
[0059] In accordance with the present invention, an isolated
nucleic acid molecule is provided that encodes a novel regulator of
ABA-mediated stomata aperture control. This nucleic acid molecule
is referred to herein as AAPK ("ABA-activated protein kinase"). Its
manner of regulating ABA-mediated stomata aperture control is novel
and interesting. When the functional product of the gene is
eliminated or specifically altered in a plant, the plant exhibits
decreased sensitivity to ABA-induced stomatal closure, but without
changing the plant's responses to stomatal closure induced by
darkness or CO.sub.2. In addition, the loss of function of AAPK had
no effect on ABA-mediated inhibition of stomatal opening. While not
intending to limit the present invention by describing one possible
mechanism of action of AAPK, it may be that AAPK functions as a
negative regulator of ABA-mediated stomatal aperture control, and
resultant transpiration and gas exchange.
[0060] AAPK was selected as an important target for cloning firstly
because its ABA-activated phosphorylation activity is specific to
guard cells. Secondly, AAPK was selected because it was not
activated by other stoma-closing factors such as CO.sub.2 or
darkness, making it a potential guard cell-specific, ABA response
regulator of stomatal closure. Furthermore, since maintaining
control of transpirational water loss and gas exchange is of a
vital and fundamental nature to plants, AAPK is likely to be a
highly conserved function among all plant species.
[0061] The AAPK cDNA was isolated from a Vicia faba cDNA library by
using degenerate primers created based on peptide sequence data.
The degenerate primers were used in reverse transcriptase--PCR to
generate a 310 bp probe from guard cell RNA. The probe was then
used to screen a V. faba guard cell cDNA library. Based on analysis
of the probes, the AAPK gene product appears to be a significant
protein kinase in the guard cells, inasmuch as other protein
kinases were not identified despite the fact that the probe was
homologous to domains common to other protein kinase family
members. Sequence analysis of this cDNA revealed the nucleic acid
sequence of SEQ ID NO:1, and a predicted polypeptide sequence
having SEQ ID NO:2. The deduced amino acid sequence was compared to
PKABA1 (SEQ ID NO:10), the expressed product of an ABA-induced
transcript from wheat (Anderberg & Walker-Simmons, Proc. Natl.
Acad. Sci. USA 89: 10183-10187, 1992). PKABA1 is a known protein
kinase with conserved regions common to this family of kinases.
Comparison with PKABA1 revealed that AAPK also possesses these
conserved kinase domains.
[0062] Many diverse protein kinases are involved in cascading
cellular signal transduction; however the kinase domain is highly
conserved in all protein kinases. The AAPK protein sequence
contains high similarity to a large number of protein kinases, as
revealed, for example, by the alignment of plant protein kinases
shown in FIG. 1. The functional specialization that allows these
kinases to operate in specific signal transduction pathways lies
both in the kinase domain and non-kinase domains. The Vicia faba
AAPK kinase protein (SEQ ID NO:2) displays significant similarity
to PKABA1 (SEQ ID NO:10). While the similarity is highest in the
putative kinase domains, there are several regions where the two
proteins are less different from one another. PKABA is expressed
from an ABA-induced transcript, but it has not been shown to
possess the ABA-activated protein kinase activity of AAPK,
suggesting that it plays a different role.
[0063] As described in Example 2, genomic screening of an
Arabidopsis library and GenBank database screening using the SEQ ID
NO:1 cDNA reveals that the Vicia faba AAPK is most similar to the
proteins (SEQ ID NOS: 4, 5 and 7) encoded by the Arabidopsis genes
having Genbank Accession Numbers L05561 and L05562, and Arabidopsis
protein having Accession No. CAA19877, respectively, indicating
that these genes and proteins are clear functional homologs of
Vicia faba AAPK and its encoded protein.
[0064] An additional round of database screening was performed,
using peptide segments of SEQ ID NO:2 that were homologous to SEQ
ID NO: 4 (encoded by GenBank L05562, Arabidopsis Atpk) but
different from wheat PKABA1 (SEQ ID NO:10). These peptides were:
(1) PIMHDSDRYDF (SEQ ID NO:15), corresponding to residues 5-15 of
SEQ ID NO:2 at the amino terminus; and (2) PADLVNENIMDNQFEEPDQ (SEQ
ID NO:16), corresponding to residues 275-293 of SEQ ID NO:2 near
the carboxyl terminus. Screening with either of these peptides
corroborated the physical and database screening using the complete
sequence, identifying each the aforementioned Arabidopsis proteins.
This screening also revealed a fourth homolog, identified in the
database as Protein Kinase SPK-2, Accession No. S56718 (SEQ ID
NO:8). It is possible that this Arabidopsis protein is the same as
the predicted protein from clone L05561.
[0065] Although the AAPK cDNA clone from Vicia faba, and homologs
from Arabidopsis are described and exemplified herein, this
invention is intended to encompass nucleic acid sequences and
proteins from other plants that are sufficiently similar to be used
instead of the Vicia faba or Arabidopsis AAPK nucleic acids and
proteins for the purposes described below. These include, but are
not limited to, allelic variants and natural mutants of AAPK, which
are likely to be found in different varieties of Vicia faba, as
well as homologs of AAPK from different species of plants. Because
such variants and homologs are expected to possess certain
differences in nucleotide and amino acid sequence, this invention
provides an isolated AAPK nucleic acid molecule having at least
about 50% (preferably 60%, more preferably 70% and even more
preferably over 80%) sequence identity in the coding regions with
the nucleotide sequence set forth as SEQ ID NOs:1, 3, 5, 7 or 9
(and, most preferably, specifically comprising the coding regions
of SEQ ID NOs:1, 3, or 6 This invention also provides isolated
polypeptide products of the open reading frames of AAPK, having at
least about 60% (preferably 70%, 75%, 80% or greater) sequence
identity with the amino acid sequences of SEQ ID NOS: 2, 4, 5, 7 or
9. Because of the natural sequence variation likely to exist among
AAPK genes, one skilled in the art would expect to find up to about
30-40% nucleotide sequence variation, while still maintaining the
unique properties of the AAPK nucleic acid molecules and encoded
polypeptides of the present invention. Such an expectation is due
in part to the degeneracy of the genetic code, as well as to the
known evolutionary success of conservative amino acid sequence
variations, which do not appreciably alter the nature of the
encoded protein. Accordingly, such variants and homologs are
considered substantially the same as one another and are included
within the scope of the present invention. Within the parameters of
sequence identity and similarity set forth above, AAPKs from any
plant species are considered part of the present invention. Such
plant species include dicotyledenous and monocotyledenous flowering
plants, as well as any other plant that possesses stomata. Of
particular importance to the invention are AAPKs from agronomically
or horticulturally important plant species, including maize, wheat,
rye, oats, barley, rice, sorghum, soy and other beans, alfalfa,
sunflower, canola, lawn and turfgrasses, tobacco, aster, zinnia,
chrysanthemum, beet, carrot, cruciferous vegetables, cucumber,
grape, pea, potato, rutabaga, tomato, tomatillo and turnip, to name
a few.
[0066] AAPK nucleic acid molecules of the invention include DNA,
RNA, and fragments thereof which may be single- or double-stranded.
Thus, this invention provides oligonucleotides (sense or antisense
strands of DNA or RNA) having sequences capable of hybridizing with
at least one sequence of a nucleic acid molecule encoding the
protein of the present invention. Such oligonucleotides are useful
as probes for detecting AAPK genes or transcripts.
[0067] In addition to encompassing natural mutants of AAPK, the
present invention is drawn to artificially created mutants,
produced by in vitro mutagenesis or isolated from mutagenized
plants, as described in greater detail below. These mutant AAPK
nucleic acids and their encoded proteins are integral to practicing
the methods of the invention, which involve regulating ABA-mediated
stomatal closure in plants. The present invention further
encompasses genetically modified plants having altered
transpiration and gas exchange characteristics due to the
down-regulation or up-regulation of AAPK in those plants.
[0068] The following sections set forth the general procedures
involved in practicing the present invention in all of its aspects
as summarized above. To the extent that specific materials are
mentioned, it is merely for purposes of illustration and is not
intended to limit the invention. Unless otherwise specified,
general cloning procedures, such as those set forth in Sambrook et
al., Molecular Cloning, Cold Spring Harbor Laboratory (1989)
(hereinafter "Sambrook et al.") or Ausubel et al. (eds) Current
Protocols in Molecular Biology, John Wiley & Sons (2000)
(hereinafter "Ausubel et al.") are used.
[0069] A. Preparation of AAPK Nucleic Acids, Proteins, Antibodies,
AAPK Mutants and Transgenic Plants
[0070] Preparation of AAPK Nucleic Acid Molecules. AAPK nucleic
acid molecules of the invention may be prepared by two general
methods: (1) they may be synthesized from appropriate nucleotide
triphosphates, or (2) they may be isolated from biological sources.
Both methods utilize protocols well known in the art.
[0071] The availability of nucleotide sequence information, such
SEQ ID NOS: 1, 3 and 6, enables preparation of an isolated nucleic
acid molecule of the invention by polynucleotide synthesis.
Synthetic oligonucleotides may be prepared by the phosphoramadite
method employed in the Applied Biosystems 38A DNA Synthesizer or
similar devices. The resultant construct may be purified according
to methods known in the art, such as high performance liquid
chromatography (HPLC). Long, double-stranded polynucleotides, such
as a DNA molecule of the present invention, must be synthesized in
stages, due to the size limitations inherent in current
oligonucleotide synthetic methods. Thus, for example, a long
double-stranded molecule may be synthesized as several smaller
segments of appropriate complementarity. Complementary segments
thus produced may be annealed such that each segment possesses
appropriate cohesive termini for attachment of an adjacent segment.
Adjacent segments may be ligated by annealing cohesive termini in
the presence of DNA ligase to construct an entire long
double-stranded molecule. A synthetic DNA molecule so constructed
may then be cloned and amplified in an appropriate vector.
[0072] Modified (i.e., "mutant") nucleic acid molecules of the
invention also may be synthesized as described above. In this
embodiment, the desired alteration is simply programmed into the
synthetic scheme. In another embodiment, an unaltered synthetic
nucleic acid molecule is manufactured, and subsequently altered by
site-directed mutagenesis.
[0073] Nucleic acid molecules encoding the AAPK protein may be
isolated from V. faba, Arabidopsis or any other plant of interest
using methods well known in the art. It will be appreciated that
such methods may be used to screen libraries of mutant plants as
well as wild-type plants. In order to isolate AAPK-encoding nucleic
acids from plants other than V. faba, or Arabidopsis,
oligonucleotides designed to match the nucleic acids encoding the
V. faba or Arabidopsis AAPK protein may be used with cDNA or
genomic libraries from the desired species. If the AAPK gene from a
species is desired, the genomic library is screened. Alternately,
if the protein coding sequence is of particular interest, the cDNA
library is screened. In positions of degeneracy, where more than
one nucleic acid residue could be used to encode the appropriate
amino acid residue, all the appropriate nucleic acids residues may
be incorporated to create a mixed oligonucleotide population, or a
neutral base such as inosine may be used. Such degenerate libraries
also may be customized for the codon preference of the plant
species to be screened. The strategy of oligonucleotide design is
well known in the art (see Ausbel et al., Sambrook et al.).
[0074] In another embodiment, known AAPK sequences may be used in
"data mining" to screen databases for homologous sequences, as is
well known in the art and exemplified herein.
[0075] Alternatively, PCR (polymerase chain reaction) primers may
be designed by the above method to encode a portion a known AAPK
protein, and these primers used to amplify nucleic acids from
isolated cDNA or genomic DNA. In a preferred embodiment, the
oligonucleotides used to isolate AAPK nucleic acids are designed to
encode sequences conserved among AAPKs, but not between AAPK and
other kinases (e.g., the PKABA1 protein kinase family), as
described above.
[0076] In accordance with the present invention, nucleic acids
having the appropriate sequence homology with a known AAPK nucleic
acid molecule may be identified by using hybridization and washing
conditions of appropriate stringency. For example, hybridizations
may be performed, according to the method of Sambrook et al. (1989,
supra), using a hybridization solution comprising: 5.times.SSC,
5.times. Denhardt's reagent. 1.0% SDS, 100 .mu.g/ml denatured,
fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to
50% formamide. Hybridization is carried out at 37-42.degree. C. for
at least six hours. Following hybridization, filters are washed as
follows: (1) 5 minutes at room temperature in 2.times.SSC and 1%
SDS; (2) 15 minutes at room temperature in 2.times.SSC and 0.1%
SDS; (3) 30 minutes-1 hour at 37.degree. C. in 1.times.SSC and 1%
SDS; (4) 2 hours at 42-65.degree. in 1.times.SSC and 1% SDS,
changing the solution every 30 minutes.
[0077] One common formula for calculating the stringency conditions
required to achieve hybridization between nucleic acid molecules of
a specified sequence homology (Sambrook et al.) is:
T.sub.m=81.5 EC+16.6 Log [Na+]+0.41(% G+C)-0.63 (%
formamide)-600/@bp in duplex
As an illustration of the above formula, using [N+]=[0.368] and 50%
formamide, with GC content of 42% and an average probe size of 200
bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA duplex
decreases by 1-1.5.degree. C. with every 1% decrease in homology.
Thus, targets with greater than about 75% sequence identity would
be observed using a hybridization temperature of 42.degree. C. In a
preferred embodiment, the hybridization is at 37.degree. C. and the
final wash is at 42.degree. C., in a more preferred embodiment the
hybridization is at 42.degree. C. and the final wash is at
50.degree. C., and in a most preferred embodiment the hybridization
is at 42.degree. C. and final wash is at 65.degree. C., with the
above hybridization and wash solutions. Conditions of high
stringency include hybridization at 42.degree. C. in the above
hybridization solution and a final wash at 65.degree. C. in
0.1.times.SSC and 0.1% SDS for 10 minutes.
[0078] Nucleic acids of the present invention may be maintained as
DNA in any convenient cloning vector. In a preferred embodiment,
clones are maintained in plasmid cloning/expression vector, such as
pBluescript (Stratagene, La Jolla, Calif.), which is propagated in
a suitable E. coli host cell.
[0079] Preparation of Polypeptides and Antibodies. AAPK
polypeptides may be prepared in a variety of ways, according to
known methods. The availability of nucleic acid molecules encoding
the polypeptides enables synthesis of the proteins by known
methods, or production of the proteins using in vitro expression
methods known in the art. For example, a cDNA or gene may be cloned
into an appropriate in vitro transcription vector, such a pSP64 or
pSP65 for in vitro transcription, followed by cell-free translation
in a suitable cell-free translation system, such as wheat germ or
rabbit reticulocytes. In vitro transcription and translation
systems are commercially available, e.g., from Promega Biotech,
Madison, Wis. or BRL, Rockville, Md. The pCITE in vitro translation
system (Novagen) also may be utilized.
[0080] According to a preferred embodiment, larger quantities of
the proteins may be produced by expression in a suitable
procaryotic or eucaryotic system. For example, part or all of a DNA
molecule, such as the coding portion of SEQ ID NOS: 1, 3 or 6, or
appropriate complementary sequences, may be inserted into a plasmid
vector adapted for expression in a bacterial cell (such as E. coli)
or a yeast cell (such as Saccharomyces cerevisiae), or into a
baculovirus vector for expression in an insect cell. Such vectors
comprise the regulatory elements necessary for expression of the
DNA in the host cell, positioned in such a manner as to permit
expression of the DNA in the host cell. Such regulatory elements
required for expression include promoter sequences, transcription
initiation sequences and, optionally, enhancer sequences.
[0081] The AAPK polypeptides produced by gene expression in a
recombinant procaryotic or eucyarotic system may be purified
according to methods known in the art. In a preferred embodiment, a
commercially available expression/secretion system can be used,
whereby the recombinant protein is expressed and thereafter
secreted from the host cell, to be easily purified from the
surrounding medium. If expression/secretion vectors are not used,
an alternative approach involves purifying the recombinant protein
by affinity separation, such as by immunological interaction with
antibodies that bind specifically to the recombinant protein. Such
methods are commonly used by skilled practitioners.
[0082] AAPK proteins, prepared by the aforementioned methods, may
be analyzed according to standard procedures. Methods for analyzing
the functional activity of kinases are well known to persons
skilled in the art. Alternatively, the function of the kinase in
stomatal closure may be analyzed, as described in greater detail
below and in Example 1.
[0083] The present invention also provides antibodies that are
immunologically specific to the AAPK of the invention. Polyclonal
antibodies may be prepared according to standard methods. In a
preferred embodiment, monoclonal antibodies are prepared, which are
specific to various epitopes of the protein. Monoclonal antibodies
may be prepared according to general methods of Kohler and
Milstein, following standard protocols. Polyclonal or monoclonal
antibodies that are immunologically specific for the AAPK can be
utilized for identifying and purifying AAPK from V. faba and other
species. For example, antibodies may be utilized for affinity
separation of proteins for which they are specific or to quantify
the protein. Antibodies may also be used to immunoprecipitate
proteins from a sample containing a mixture of proteins and other
biological molecules.
[0084] Mutants and Transgenic Plants. Example 1 describes a
synthetic mutant, AAPK(K43A) in Vicia faba, which displays
insensitivity to ABA-induce stomatal closure due to the loss of
function of AAPK. Any plant may be transgenically engineered to
display a similar phenotype. This approach is particularly
appropriate to plants with high ploidy numbers, including but not
limited to wheat.
[0085] These synthetic null mutant are created by a expressing a
mutant form of the AAPK protein to create a "dominant negative
effect". While not limiting the invention to any one mechanism,
this mutant AAPK protein competes with wild-type AAPK protein for
interacting proteins in the transgenic plant, or poisons an AAPK
multimeric complex. By over-producing the mutant form of the
protein, the signaling pathway used by the wild-type AAPK protein
can be effectively blocked. Examples of this type of "dominant
negative" effect are well known for both insect and vertebrate
systems (Radke et al, 1997, Genetics 145:163-171; Kolch et al.,
1991, Nature 349:426-428). In a preferred embodiment, the mutant
protein is produced by mutating the coding sequence of AAPK
corresponding to residues in the active site. In a particularly
preferred embodiment, the coding sequence corresponding to the
lysine residue at position 43 is mutated to code for a different,
preferably non-similar, amino acid residue, for example,
alanine.
[0086] A second kind of synthetic null mutant can be created by
inhibiting the translation of the AAPK mRNA by
"post-transcriptional gene silencing". The AAPK gene from the
species targeted for down-regulation, or a fragment thereof, may be
utilized to control the production of the encoded protein.
Full-length antisense molecules or antisense oligonucleotides are
used that are targeted to specific regions of the AAPK-encoded RNA
that are critical for translation. The use of antisense molecules
to decrease expression levels of a pre-determined gene is known in
the art. Antisense molecules may be provided in situ by
transforming plant cells with a DNA construct which, upon
transcription, produces the antisense RNA sequences. Such
constructs can be designed to produce full-length or partial
antisense sequences. This gene silencing effect can be enhanced by
transgenically over-producing both sense and antisense RNA of the
gene coding sequence so that a high amount of dsRNA is produced
(for example see Waterhouse et al., 1998, PNAS 95:13959-13964). In
a preferred embodiment, part or all of the AAPK coding sequence
antisense strand is expressed by a transgene. In a particularly
preferred embodiment, hybridizing sense and antisense strands of
part or all of the AAPK coding sequence are transgenically
expressed.
[0087] A third type of synthetic null mutant can also be created by
the technique of "co-suppression". Plant cells are transformed with
a copy of the endogenous gene targeted for repression. In many
cases, this results in the complete repression of the native gene
as well as the transgene. In a preferred embodiment, the AAPK gene
from the plant species of interest is isolated and used to
transform cells of that same species.
[0088] Transgenic plants can also be created that have enhanced
AAPK activity. This can be accomplished by transforming plant cells
with a transgene that expresses part or all of the AAPK coding
sequence, or a sequence that encodes the either the AAPK protein or
a protein functionally similar to it. In a preferred embodiment,
the complete AAPK coding sequence is transgenically over-expressed.
In another embodiment, the coding sequence corresponding to the
kinase domain of AAPK is over-expressed.
[0089] Transgenic plants with one of the transgenes mentioned above
can be generated using standard plant transformation methods known
to those skilled in the art. These include, but are not limited to,
Agrobacterium vectors, polyethylene glycol treatment of
protoplasts, biolistic DNA delivery, UV laser microbeam, gemini
virus vectors, calcium phosphate treatment of protoplasts,
electroporation of isolated protoplasts, agitation of cell
suspensions in solution with microbeads coated with the
transforming DNA, agitation of cell suspension in solution with
silicon fibers coated with transforming DNA, direct DNA uptake,
liposome-mediated DNA uptake, and the like. Such methods have been
published in the art. See, e.g., Methods for Plant Molecular
Biology (Weissbach & Weissbach, eds., 1988); Methods in Plant
Molecular Biology (Schuler & Zielinski, eds., 1989); Plant
Molecular Biology Manual (Gelvin, Schilperoort, Verma, eds., 1993);
and Methods in Plant Molecular Biology--A Laboratory Manual
(Maliga, Klessig, Cashmore, Gruissem & Varner, eds., 1994).
[0090] The method of transformation depends upon the plant to be
transformed. Agrobacterium vectors are often used to transform
dicot species. Agrobacterium binary vectors include, but are not
limited to, BIN19 (Bevan, 1984) and derivatives thereof, the pBI
vector series (Jefferson et al., 1987), and binary vectors pGA482
and pGA492 (An, 1986) For transformation of monocot species,
biolistic bombardment with particles coated with transforming DNA
and silicon fibers coated with transforming DNA are often useful
for nuclear transformation.
[0091] DNA constructs for transforming a selected plant comprise a
coding sequence of interest operably linked to appropriate 5'
(e.g., promoters and translational regulatory sequences) and 3'
regulatory sequences (e.g., terminators). In a preferred
embodiment, the coding region is placed under a powerful
constitutive promoter, such as the Cauliflower Mosaic Virus (CaMV)
35S promoter or the figwort mosaic virus 35S promoter. Other
constitutive promoters contemplated for use in the present
invention include, but are not limited to: T-DNA mannopine
synthetase, nopaline synthase (NOS) and octopine synthase (OCS)
promoters.
[0092] Transgenic plants expressing a sense or antisense AAPK
coding sequence under an inducible promoter are also contemplated
to be within the scope of the present invention. Inducible plant
promoters include the tetracycline repressor/operator controlled
promoter, the heat shock gene promoters, stress (e.g.,
wounding)-induced promoters, defense responsive gene promoters
(e.g. phenylalanine ammonia lyase genes), wound induced gene
promoters (e.g. hydroxyproline rich cell wall protein genes),
chemically-inducible gene promoters (e.g., nitrate reductase genes,
glucanase genes, chitinase genes, etc.) and dark-inducible gene
promoters (e.g., asparagine synthetase gene) to name a few.
[0093] Tissue specific and development-specific promoters are also
contemplated for use in the present invention. Examples of these
included, but are not limited to: the ribulose bisphosphate
carboxylase (RuBisCo) small subunit gene promoters or chlorophyll
a/b binding protein (CAB) gene promoters for expression in
photosynthetic tissue; the various seed storage protein gene
promoters for expression in seeds; and the root-specific glutamine
synthetase gene promoters where expression in roots is desired.
Although the AAPK gene of the preferred embodiments taught with
this invention are specifically expressed in guard cells, this in
no way limits the application of this invention to any specific
tissue or development phase, but rather represents only the
particular embodiments taught herein.
[0094] The coding region is also operably linked to an appropriate
3' regulatory sequence. In a preferred embodiment, the nopaline
synthetase polyadenylation region (NOS) is used. Other useful 3'
regulatory regions include, but are not limited to the octopine
(OCS) polyadenylation region.
[0095] Using an Agrobacterium binary vector system for
transformation, the selected coding region, under control of a
constitutive or inducible promoter as described above, is linked to
a nuclear drug resistance marker, such as kanamycin resistance.
Other useful selectable marker systems include, but are not limited
to: other genes that confer antibiotic resistances (e.g.,
resistance to hygromycin or bialaphos) or herbicide resistance
(e.g., resistance to sulfonylurea, phosphinothricin, or
glyphosate).
[0096] Plants are transformed and thereafter screened for one or
more properties, including the lack of AAPK protein, AAPK mRNA, or
altered stomatal aperture responses to ABA treatment. It should be
recognized that the amount of expression, as well as the
tissue-specific pattern of expression of the transgenes in
transformed plants can vary depending on the position of their
insertion into the nuclear genome. Such positional effects are well
known in the art. For this reason, several nuclear transformants
should be regenerated and tested for expression of the
transgene.
[0097] Transgenic plants that exhibit one or more of the
aforementioned desirable phenotypes can be used for plant breeding,
or directly in agricultural or horticultural applications. Plants
containing one transgene may also be crossed with plants containing
a complementary transgene in order to produce plants with enhanced
or combined phenotypes.
[0098] An alternative to the transgenic approach described above is
the screening of populations of plant mutants of a variety of
species, from which AAPK mutants can be isolated. Such populations
can be made by chemical mutagenesis, radiation mutagenesis, and
transposon or T-DNA insertion, as is well known in the art. In a
preferred embodiment, the mutants would be null mutants having a
phenotype comprising reduced or substantially absent stomatal
closure in response to abscisic acid, but no reduction in stomatal
closure response to darkness or CO.sub.2. In yet another preferred
embodiment, mutant are screened for the phenotypes related to
overproduction of the AAPK gene product and/or increased
sensitivity to ABA-induced stomatal closure.
[0099] The nucleic acids of the invention can be used to isolate or
create AAPK mutants in a selected species. In species such as maize
where transposon insertion lines are available, oligonucleotide
primers can be designed to screen lines for insertions in the AAPK
gene. Plants with transposon or T-DNA insertions in the AAPK gene
are very likely to have lost the function of the gene product.
Through breeding, a plant line may then be developed that is
homozygous for the non-functional copy or the altered copy of the
AAPK gene. The PCR primers for this purpose are designed so that
large portions of the coding sequence the AAPK gene are
specifically amplified using the sequence of the AAPK gene from the
species to be probed (see Baumann et al., 1998, Theor. Appl. Genet.
97:729-734).
[0100] Other AAPK-like mutants can be isolated from mutant
populations using the distinctive phenotype characterized in
accordance with the present invention. This approach is
particularly appropriate in plants with low ploidy numbers where
the phenotype of a recessive mutation is more easily detected. In
order to identify these mutants, the population of plants would be
exposed to abscisic acid (ABA) or analogs of the hormone. Plants
would then be screened for phenotype of the AAPK mutants: the
reduced stomatal closure in response to applied ABA, without a
reduction in stomatal response to darkness or CO.sub.2. That the
phenotype is caused by an AAPK mutation is then established by
molecular means well known in the art.
[0101] It will be appreciated that any of the aforementioned
transformation or mutagenesis techniques may be applied to any
selected plant species. Such species include, but are not limited
to, agronomically important crop plants such as maize, wheat, rice,
rye, oats, barley, soy and other beans, sorghum, sunflower, canola,
tobacco and alfalfa; vegetable and fruit crop plants such as beet,
carrot, cruciferous vegetables, cucumber, grape, pea, potato,
rutabaga, tomato, tomatillo and turnip; and horticulturally
important plants such as aster, begonia, chrysanthemum, clover,
lawn and turf grasses, mint and other herbs, and zinnia.
[0102] B. Uses of AAPK Nucleic Acids, Proteins, Antibodies, AAPK
Mutants and Transgenic Plants
[0103] Nucleic Acid Molecules. AAPK nucleic acids may be used for a
variety of purposes in accordance with the present invention. DNA,
RNA, or fragments thereof, may be used as probes to detect the
presence and/or expression of AAPK genes. Methods in which AAPK
nucleic acids may be utilized as probes for such assays include,
but are not limited to: (1) in situ hybridization; (2) Southern
hybridization (3) Northern hybridization; and (4) assorted
amplification reactions such as polymerase chain reactions
(PCR).
[0104] The AAPK nucleic acids of the invention may also be utilized
as probes to identify related genes from other plant species. As is
well known in the art, hybridization stringencies may be adjusted
to allow hybridization of nucleic acid probes with complementary
sequences of varying degrees of homology. As described above, AAPK
nucleic acids may be used to advantage to produce large quantities
of substantially pure AAPK, or selected portions thereof. The AAPK
nucleic acids can be used to identify and isolate other putative
members of this novel ABA-mediated stomatal aperture control signal
cascade in vivo. A yeast two hybrid system can be used to identify
proteins that physically interact with the AAPK protein, as well as
isolate their nucleic acids. In this system, the sequence encoding
the protein of interest is operably linked to the sequence encoding
half of a activator protein. This construct is used to transform a
yeast cell library which has been transformed with DNA constructs
that contain the coding sequence for the other half of the
activator protein operably linked to a random coding sequence from
the organism of interest. When the protein made by the random
coding sequence from the library interacts with the protein of
interest, the two halves of the activator protein are physically
associated and form a functional unit that activates the reporter
gene. In accordance with the present invention, all or part of the
AAPK coding sequence may be operably linked to the coding sequence
of the first half of the activator, and the library of random
coding sequences may be constructed with cDNA from V. faba and
operably linked to the coding sequence of the second half of the
activator protein. Several activator protein/reporter genes are
customarily used in the yeast two hybrid system. In a preferred
embodiment, the bacterial repressor LexA DNA-binding domain and the
Gal4 transcription activation domain fusion proteins associate to
activate the LacZ reporter gene (see Clark et al., 1998, PNAS
95:5401-5406). Kits for the two hybrid system are also commercially
available from Clontech, Palo Alto Calif., among others.
[0105] In a preferred embodiment, interaction cloning is used
identify proteins that physically interact with the AAPK protein
and to isolate the nucleic acids encoding them. In this method, a
cDNA expression library is screened for proteins that interact with
the AAPK catalytic domain, or other selected domains that might be
involved in protein-protein interactions. This is done using a
filter binding assay and a labeled peptide comprising the putative
interacting site. Positive clones are then purified, amplified if
necessary, and characterized.
[0106] Proteins and Antibodies. The AAPK proteins of the present
invention can be used to identify molecules with binding affinity
for AAPK, which are likely to be novel participants in this
resistance pathway. In these assays, the known protein is allowed
to form a physical interaction with the unknown binding
molecule(s), often in a heterogenous solution of proteins. The
known protein in complex with associated molecules is then
isolated, and the nature of the associated protein(s) and/or other
molecules is determined.
[0107] AAPK may also be generated as part of a fusion protein with
one or more other proteins, for example with a green fluorescent
protein (GFP). Such fusion products may have utility from either or
each part of the fusion molecule. For example, the easy detection
of their presence is provided by the GFP moiety, while the specific
kinase activity is retained by the AAPK moiety. Additionally they
may allow convenient use of commercially available antibodies
specific to the fused portion (e.g antiGFP antibodies are readily
available.)
[0108] Antibodies that are immunologically specific for AAPK may be
utilized in affinity chromatography to isolate the AAPK protein, to
identify or quantify the AAPK protein utilizing techniques such as
western blotting and ELISA, or to immuno-precipitate AAPK from a
sample containing a mixture of proteins and other biological
materials. The immuno-precipitation of AAPK is particularly
advantageous when utilized to isolate affinity binding complexes of
AAPK, as described above.
[0109] Mutants and Transgenic Plants. The AAPK mutants of the
invention display altered sensitivity to ABA-induced stomatal
closure, and therefore can be used to improve crop and
horticultural plant species. The AAPK mutants taught in this
invention are particularly valuable in that the mutation is very
specific. The altered sensitivity is found only in guard cells, and
stomatal closure by other means such as darkness or CO.sub.2 is
unaffected. Such mutants will therefore be particularly useful in
crop and horticultural varieties in which reduction of moisture
content is important. Examples of such crops include but are not
limited to cereal grains such as corn, wheat, rye, oats, barley,
and rice, soybeans and other beans, as well as other products such
as hay and commercial seed. In most of these cases failure to
adequately dry the crop due to weather or other conditions results
in substantial losses. In other cases including but not limited to
tobacco, dried fruits such as raisins and prunes, nuts, coffee,
tea, cocoa, and many ornamental goods, the produce needs to be
dried immediately after harvest prior and to use. In these cases
again, the mutants of this invention may be of tremendous value to
growers who could accelerate or control the rate of crop
drying.
[0110] The AAPK mutants exhibit a decreased induction by ABA of
normal stomatal closure. They therefore have influence over
transpirational water loss in plants. It is therefore contemplated
that in addition to the specific applications mentioned heretofore,
these mutants will have myriad applications to other important
plant problems especially in irrigated crops or other crops where
water and yield are delicately balanced.
[0111] It is also trivial to one skilled in the art to extend this
invention to the production of mutants with increased sensitivity
to ABA-induced stomatal closure. These mutants are useful for a
variety agronomic purposes. It is clear that such mutants would
keep the stomatal aperture small, and would therefore experience
reduced transpirational water loss. Such mutants can be used to
help enhance tolerance to water stress or drought conditions. Such
mutants could be the result of active site changes or modifications
which allow them to respond to lower concentrations of ABA, or they
could be the result of mutations in genes in a common regulatory
pathway. Such mutants could also be the result of overexpression of
the AAPK gene product via a transgenic modification such that
expression of APK is driven by an inducible promoter, a strong,
highly active constitutive promoter, or by increasing the copy
number of the gene in the plant. These approaches are all
conceptually simple to one skilled in the art, and other approaches
may be preferred for particular embodiments.
[0112] The AAPK mutants of the invention can be used to identify
and isolate additional members of this ABA-regulation of
transpiration pathway. Mutations that, when combined with AAPK
mutations, suppress the mutant phenotype, are likely to interact
directly with AAPK, or to compensate in some significant indirect
way for the loss of AAPK function. Since AAPK is known to be a
protein kinase with both autophosphorylation and substrate
phosphorylation, there are opportunities to identify other
important members of the ABA signal cascade using the mutants of
this present invention.
[0113] The transgenic plants of the invention are particularly
useful in conferring the AAPK phenotype to many different plant
species. In this manner, a host of plant species with enhanced
disease resistance can be easily made, to be used as breeding lines
or directly in commercial operations. Such plants can have uses as
crop species, or for ornamental use.
[0114] A plant that has had functional AAPK transgenically depleted
will exhibit the same altered sensitivity to ABA-induced stomatal
closure as AAPK mutants. It is therefore contemplated that
transgenic AAPK-phenotype plants will be used with in the same
aforementioned manner as the AAPK mutants. A transgenic approach is
advantageous because it allows AAPK-phenotype plants to be created
quickly, without time-consuming mutant generation, selection, and
back-crossing.
[0115] A plant that has had functional AAPK increased may have
enhanced sensitivity to ABA-induced stomatal closure compared to
wild-type plants. Plants with enhanced sensitivity to ABA-induced
stomatal closure will be extremely valuable to agriculture and
horticulture by allowing plants to better tolerate periods of
restricted water or drought. Additionally, such mutants may provide
the advantage of allowing produce to retain water as long as
possible. For many fruits, vegetables and flowers, including cut
flowers, it would be advantageous to minimize water loss during the
harvest, transport and distribution. Retail customers too would
benefit from the extended shelf-life of such fruits, vegetables and
flowers which would remain fresher for longer periods of time.
[0116] The following examples are provided to describe the
invention in greater detail. They are intended to illustrate, not
to limit, the invention.
EXAMPLE 1
Cloning and Characterization of ABA-Activated Protein
Kinase-Encoding cDNA from Guard Cells
[0117] This example describes the cloning and characterization of a
Vicia faba complementary DNA, AAPK, encoding a guard cell-specific
ABA-activated serine-threonine protein kinase (AAPK).
Methods
[0118] Isolation and Identification of ABA-Activated Protein Kinase
(AAPK). Guard cell protoplasts (2.times.10.sup.6; 99.6% pure) were
prepared from Vicia faba. Protoplasts were treated with either
darkness, ABA or elevated CO.sub.2 concentrations prior to protein
isolation. Protoplast proteins were extracted and subjected to
2-dimensional gel electrophoresis. Separation was via 12%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Kinase
autophosphorylation activity of subsequently renatured proteins was
detected by established methods.
[0119] The AAPK protein was excised from the 2-D gels (first
dimension, nondenaturing PAGE) after digestion with trypsin. The
AAPK peptides generated by trypsin digestion were subjected to
peptide sequencing by tandem mass spectrometry on a Finnigan LCQ
quadrupole ion trap mass spectrometer.
[0120] Cloning of ABA-Activated Protein Kinase (AAPK). The gene
encoding AAPK was cloned by screening a V. faba cDNA library with
probes constructed based on peptide sequence data obtained from the
mass spectrometry analysis.
[0121] Degenerate DNA primers whose design was based on sequences
conserved between subdomain II of the protein kinase ABA1 (PKABA1)
protein kinase subfamily and the AAPK sequence corresponding to
protein kinase subdomain VIb, were synthesized. The degenerate
primers {5'-TTGC(C/T)(A/G)T(G/C) AA(A/G)TACATCGAA-3' (SEQ ID NO:17)
(forward primer, located in subdomain II),
5'-CCATC(C/T)A(A/G)NAGNGT(A/G)TTTTC-3' (SEQ ID NO:18) (reverse
primer, located in subdomain VIb) where N=A+G+C+T} were used for
reverse transcription-polymerase chain reaction (RT-PCR) using as
template total RNA from guard cells. of V. faba. The PCR product
was labelled with {.sup.32P}dCTP.
[0122] This .sup.32P-labelled probe was then used to screen a V.
faba guard cell cDNA library in .lamda.-Zap II. A full length cDNA
of the appropriate size to encode the AAPK was obtained. The cDNA
was sequenced in both directions.
[0123] Total RNA was isolated from purified guard cells, mesophyll
cells, flowers, leaves and seeds of V. faba. Northern analysis was
performed by standard methods. The probe was the .sup.32P-labelled
BglII-Csp45I fragment of the AAPK cDNA clone. This probe includes
the sequence encoding the relatively unique AAPK amino terminal
region and part of the 3' untranslated region of the cDNA.
[0124] Functional Analysis of the AAPK Gene. Functional analysis of
the AAPK gene product was complicated by the observation that
ABA-activation of the AAPK does not occur in vitro. Prior treatment
of intact guard cells with ABA was required to elicit active AAPK
upon extraction. In light of this apparent requirement for an
intact cellular signal system, DNA constructs were created to
facilitate the analysis of expression and activity of AAPK.
[0125] Creation of AAPK Mutants and Hybrid Expression DNA
Constructs. A construct encoding a green fluorescent protein
(GFP)-tagged AAPK was made. This construct, pAAPK-GFP, was created
by amplifying the AAPK coding sequence from the AAPK cDNA and
inserting the amplified coding sequence downstream of the 35S
promoter and upstream of, and in-frame with, the GFP coding
sequence in the GFP expression vector, pGFP. The amplification was
performed via PCR with the primers
5'-GAATCTCCACTACGACGCCGTTTACTTCCC-3' (SEQ ID NO:19) and
5'-CCGTGCAACCATGGATATGGCATATACAAT-3' (SEQ ID NO:20). NcoI was used
for digestion and insertion.
[0126] Another DNA construct, pAAPK(K43A)-GFP was created. This
construct contained a site-directed mutation of AAPK, such that the
coding sequence for a highly conserved lysine residue (Lys.sup.43),
believed to be in the ATP-binding site of the kinase AAPK, was
specifically modified to encode an alanine residue instead of the
lysine. Such mutations have been shown to yield kinases with
reduced or absent catalytic activity.
[0127] Analysis of AAPK Mutants and Hybrid Expression DNA
Constructs for ABA-Activated Protein Kinase Activity.
1.5.times.10.sup.7 V. faba guard cell protoplasts were transfected
with either the vector, pGFP, or the constructs, pAAPK-GFP or
pAAPK(K43A)-GFP by polyethylene glycol (PEG)-mediated DNA transfer.
After uptake and expression, protoplasts were lysed and recombinant
protein was immunoprecipitated with anti-GFP peptide antibodies
(Clontech) and protein A-Sepharose CL-4B (Amersham Pharmacia
Biotech). Immunoprecipitated proteins were assayed for kinase
activity using histone III-S (Sigma) as substrate.
[0128] Analysis of AAPK Mutants for Stomatal Aperture Changes and
Anion Channel Activation. V. faba leaves were biolistically
transformed with the pGFP, pAAPK-GFP, or pAAPK(K43A)-GFP
constructs. The V. faba leaves were bombarded with gold particles
(Bio-Rad) coated with one of the DNA constructs. Bombardment was
via a particle delivery system 1000/He (Bio-Rad) as described. (J.
Marc et al., 1998, Plant Cell 10:1927).
[0129] Abaxial epidermal peels were isolated and the transformed
guard cells were assayed for ABA-mediated prevention of stomatal
opening and for stomatal closure stimulated by ABA, CO.sub.2 or
darkness. Conditions were as in Assman, S., and Baskin, T. (1998)
J. Exp. Bot. 49:163 except that the incubation solution was 10 mM
MES, 30 mM KCl, pH=6.1, with or without 50 .mu.M {.+-.}
cis,trans-ABA. For closure experiments, the transformed leaves were
illuminated with 0.20 mmol m.sup.-2 s.sup.-1 white light for 2.5 h
to open the stomata. The abaxial epidermal peels were placed in
incubation solution and treated with either darkness, 25 .mu.M
{.+-.} cis,trans-ABA or with 700 ppm CO.sub.2 for 1 h.
[0130] Anion channel activation was measured in guard cell
protoplasts. Whole-cell patch-clamp experiments were performed
according to established methods. Pipette solution contained 100 mM
KCl, 50 mM tetramethylammonium, 2 mM MgCl, 6.7 mM
EGTA-(Tris).sub.2, 3.35 mM CaCl.sub.2, 10 mM HEPES, pH=7.1 (Tris)
and 5 mM Mg-ATP. Bath solution contained 40 mM CaCl.sub.2, 2 mM
MgCl and 10 mM MES-Tris pH5.6. Osmolalities were adjusted with
sorbitol to 500 mosmol/kg (in the pipette) or 470 mosmol/kg (in the
bath). Protein kinase inhibitor K-252a (Calbiochem) was prepared as
a stock solution at 2 mM in dimethyl sulfoxide (DMSO).
Results and Discussion
[0131] The Vicia faba ABA-Activated Protein Kinase (AAPK) is a 48
kDa Protein. AAPK was identified as a 48 kDa ABA-dependent and
Ca.sup.2+-independent autophosphorylation spot with the in-gel
kinase assay. The peptide fragment sequence information obtained is
provided in FIG. 1. Two sequenced AAPK peptides had similarity to
the protein kinase ABA1 (PKABA1) protein kinase subfamily in
subdomains I and VIb. PKABA1 is transcriptionally up-regulated by
ABA.
[0132] Cloning of a Guard-Cell-Specific AAPK Gene Encoding
ABA-Activated Protein Kinase (AAPK). The RT-PCR of total guard cell
RNA using the degenerate primers yielded a 310 base pair sequence
which encoded the peptides sequences from the AAPK and also encoded
a sequence similar to that of the PKABA1 subfamily from subdomains
II to VIb. A full length cDNA of the appropriate size to encode the
AAPK was obtained from the screening of the V. faba cDNA library
with this probe.
[0133] The nucleotide sequence of the full length AAPK cDNA and the
amino acid sequence of the deduced AAPK protein are given in FIG.
1. The deduced AAPK amino acid sequence shows the greatest homology
to the PKABA1 subfamily. However, the predicted sequence also has
unique regions.
[0134] Northern analysis showed that AAPK mRNA is expressed in
guard cell protoplasts but not in mesophyll cell protoplasts,
flowers, leaves, or seeds; true to the pattern of guard
cell-specificity previously observed for AAPK activity. AAPK
appears to be a single copy gene based on results from Southern
analysis; however, further analysis may reveal the presence of more
than one copy.
[0135] The in situ activation requirement for AAPK activity could
be an indication that an intact cellular signal or cascade is
required. Any discussion or explanation offered here is intended to
provide clarity and is not intended to limit the invention in any
way to one theory or avenue as to the mechanism or application.
[0136] ABA-Dependent Autophosphorylation and ABA-Activated
Substrate Phosphorylation. No histone phosphorylation was observed
by the proteins immunoprecipitated from guard cells transformed
with the control vector (pGFP) only. Phosphorylation activity of
the fusion proteins AAPK-GFP and AAPK(K43A)-GFP was also
determined. The immunoprecipitate from cells transfected with
pAAPK-GFP showed histone phosphorylation activity which was
significantly enhanced when the protoplasts were treated with ABA
prior to isolation of the proteins. The fusion product of pAAPK-GFP
showed both ABA-dependent autophosphorylation and the ABA-activated
histone phophorylation, establishing that the cloned gene indeed
encodes the observed biochemical activity of AAPK. The
immunoprecipitate from cells transfected with pAAPK(K43A)-GFP,
encoding the site-mutagenized AAPK(K43A), also showed ABA-dependent
autophosphorylation and ABA-activated histone phosphorylation
activity, however the relative levels were significantly reduced as
would be predicted from the sequence change.
[0137] ABA-Mediated Stomatal Aperture Regulation and Anion Channel
Activation in AAPK Mutants. The transformed guard cells were
identified by their green fluorescence. Transformation with
pAAPK(K43A)-GFP eliminated ABA-induced stomatal closure, but had no
effect on stomatal closure induced by CO.sub.2 or darkness.
Transformation with wild-type AAPK via the pAAPK-GFP vector had no
measurable effect on either ABA-induced stomatal closure nor on
ABA-inhibition of stomatal opening.
[0138] In V. faba guard cells, ABA activated slow anion channels.
Slow anion currents were identified by their characteristic time
dependence, their reversal potential and sensitivity to the anion
channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid. Not
only was the typical decay in anion current reversed over time in
the whole-cell configuration, but ABA also increased the anion
current magnitude. In guard cells transformed with pGFP or
pAAPK-GFP, anion currents were regulated normally (activated) by
ABA. In guard cells transformed with pAAPK(K43A)-GFP, however,
ABA-activation of anion channels was eliminated.
[0139] It is likely that the K43A mutant kinase competes with the
activity of native AAPK in a dominant negative fashion. First, the
kinase inhibitor K-252a inhibits (i) native AAPK activity, (ii)
ABA-induced stomatal closure, and (iii) ABA regulation of anion
channels in untransformed cells, implying that the channels are
indeed normally regulated by AAPK. Second, although dominant abi1-1
and abi2-1 mutations in ABI and ABI2 phosphatases confer ABA
insensitivity to both anion channel activation and stomatal
closure, recently identified recessive loss-of-function mutations
in ABI1 confer hypersensitivity to ABA. Thus, in wild-type plants
an AAPK may mediate ABA-induced anion channel activation and
stomatal closure through a phosphorylation event, while ABI1
opposes ABA action through a dephosphorylation event.
[0140] Neither wild-type nor mutant versions of recombinant AAPK
affected ABA inhibition of stomatal opening ( Table 1). ABA
inhibition of stomatal opening and ABA promotion of stomatal
closure may, therefore, employ different signaling cascades.
Alternatively, and in contrast to current theory, ABA activation of
anion channels may not be required for ABA inhibition of stomatal
opening.
[0141] Agronomically, loss of ABA-stimulated stomatal closure in
plants transformed with mutant AAPK under control of an inducible
promoter should allow accelerated and controlled desiccation of
crops that are dried before harvest or distribution. Basal levels
of ABA remain even in irrigated crops; under these conditions,
inhibition of AAPK activity should alleviate stomatal limitation of
CO.sub.2 uptake, and thus accelerate growth or increase yield.
TABLE-US-00001 TABLE 1 Overexpression of AAPK(K43A) in guard cells
inhibits ABA-induced stomatal closure. V. faba leaves were
transformed and stomatal responses measured. ABA treatment was 25
.mu.M (for closure) or 50 .mu.M (for opening) (.+-.)-cis,
trans-ABA, elevated CO.sub.2 treatment was 700 ppm CO.sub.2. Except
for the darkness treatment, peels were illuminated (0.20 mmol
m.sup.-2s.sup.-1 white light) for the duration of each treatment.
All numbers represent the change in half aperture of stomata as
measured in micrometers. ND, not determined. Numbers in parentheses
indicate sample sizes. GFP AAPK-GFP AAPK (K43A)-GFP Transformed
Untransformed Transformed Untransformed Transformed Untransformed
Closure ABA -2.52 .+-. 0.29 (36) -2.54 .+-. 0.35 (36) -2.59 .+-.
0.30 (36) -2.58 .+-. 0.24 (36) -0.36 .+-. 0.26 (56)* -2.55 .+-.
0.21 (56) Control 0.10 .+-. 0.09 (10) 0.09 .+-. 0.09 (10) 0.11 .+-.
0.10 (10) 0.12 .+-. 0.10 (24) 0.12 .+-. 0.11 (10) 0.13 .+-. 0.10
(24) CO.sub.2 ND ND ND ND -2.23 .+-. 0.44 (36) -2.31 .+-. 0.46 (36)
Control -0.09 .+-. 0.09 (10) -0.11 .+-. 0.10 (10) Darkness ND ND ND
ND -2.08 .+-. 0.40 (36) -2.08 .+-. 0.46 (36) Control 0.12 .+-. 0.11
(10) 0.13 .+-. 0.11 (10) Opening ABA 0.42 .+-. 0.22 (36) 0.45 .+-.
0.27 (36) 0.43 .+-. 0.22 (36) 0.41 .+-. 0.28 (36) 0.42 .+-. 0.17
(46) 0.44 .+-. 0.15 (46) *Significantly different from
Untransformed cells treated with ABA (P < 0.001, Student's t
test). Not significantly different from the AAPK(K43A)-GFP
transformed ABA control (P > 0.05, Student's t test).
EXAMPLE 2
Identification of AAPK Genes from Arabidopsis thaliana
[0142] Screening. Standard methods known to those skilled in the
art for screening a genomic library were used. An Arabidopsis
genomic library from CD4-8 Landsberg erecta from the Arabidopsis
Biological Resource Center at Ohio State University was used.
[0143] The probe was the Nco I-Bgl II fragment (393 base pairs) of
V. faba AAPK cDNA. The gel-purified Nco I-Bgl II fragment of AAPK
cDNA was labeled by 32P-dCTP. This probe corresponds to sequences
encoding the region from the aspartic acid residue (position 2, SEQ
ID NO:2) to the arginine residue (position 132, SEQ ID NO:2) of the
AAPK protein.
[0144] Nylon membranes containing the library were prehybridized
with 5.times.SSC, 5.times. Denhardt's solution, 1% SDS, and 0.2%
nonfat milk at 60 C for 2 hours and then hybridized in the same
solution with the labeled probe at 60 C overnight. The membranes
were washed with 2.times.SSC and 0.1% SDS twice for 15 minutes at
60 C, once in 2.times.SSC and 0.1% SDS, and once in 0.5.times.SSC
and 0.1% SDS. The membranes were exposed to X-ray films and
positive plaques were identified by autoradiography.
[0145] The positive clones were subcloned into pCR BlueScript
vector and then analyzed by DNA sequencing and compared to known
sequences to look for matches.
[0146] Data Mining. The BLAST program of the National Center for
Biological Information (NCBI) was used with the blastx option to
search the nr (nonredundant) databases of GenBank, EMBL and DDBJ
for matches with AAPK sequence data. The parameters used for the
search were: expect value, 10; matrix, BLOSUM62; filter, low
complexity.
[0147] Results. Two independent genomic clones were identified from
the screening of the Arabidopsis library. Sequencing and subsequent
database searches established that these sequences originated from
sequences embodied respectively in GenBank Accession Numbers L05562
and Protein Identification Accession Number CAA19877. The data
mining with the AAPK cDNA sequence corroborated the results
obtained through screening of the genomic library. In addition, the
database search revealed another, equally homologous, Arabidopsis
nucleic acid sequence, comprising the sequences embodied in GenBank
Accession Number L05561. The L05561 sequences correspond to a
region within the Arabidopsis BAC clone ALO31032 of chromosome
4.
[0148] Sequence comparisons revealed that the predicted polypeptide
encoded by L05562 has 75.4% identity with the amino acid sequence
encoded by the V. faba AAPK cDNA, and the nucleotide sequence has
67.9% identity to the nucleotide sequence of the V. faba. AAPK
cDNA. The predicted polypeptide encoded by CAA19877 has 77.5%
identity with the deduced V. faba AAPK amino acid sequence, and the
nucleotide sequence is 68.7% identical to the nucleic acid sequence
of V. faba AAPK cDNA. Various alignments and additional information
regarding sequence identity are set forth in FIGS. 2-4.
[0149] The present invention is not limited to the embodiments
described and exemplified above, but is capable of variation and
modification without departure from the scope of the appended
claims.
Sequence CWU 1
1
2011559DNAVicia faba 1cggcacgaga ttaaaaaggc cacaatgttg cttactctcc
aacaacaacc gtaatcctct 60cggaatctcc actacgacgc cgtttacttc cgatctctct
ccccgccgga gcagcagcca 120tggatatgcc gccgccgatc atgcacgaca
gtgaccgtta cgacttcgtt cgtgatatcg 180gatcgggaaa tttcggcgtc
gctagactca tgactgataa actcaccaaa gaccttgttg 240ctgtcaagta
catcgaacgt ggagataaga ttgatgaaaa tgttaagaga gaaataatca
300atcacaggtc tctaagacat cctaatattg ttaggtttaa ggaggtcatt
ttaacaccta 360ctcatctggc cattgtaatg gaatatgcat ctggaggaga
aatgtccgat cgaatcagca 420aagcggggcg ttttactgag gatgaggctc
gtttcttctt tcaacaactc atatccgggg 480tcagctattg tcattcaatg
caagtatgtc atcgagatct gaagttggaa aacacgttgt 540tggatggaga
cccagcactt catctgaaga tttgtgattt tggatactcc aaatcttcgg
600tgcttcattc acagccaaag tcaactgtgg gaactcctgc ttatattgct
ccagaagtac 660ttctgaagca agagtatgat ggaaagattg ccgatgtctg
gtcatgtggt gtaaccttat 720acgtgatgct agtggggtca tatccttttg
aagatcccga taatccgaag gatttccgga 780agacaattca gagggttctc
agtgtccagt attccgtacc agactttgtt caaatatctc 840ctgaatgtcg
cgacattata tcaagaatct ttgtttttga ccctgcagag agaatcacca
900ttccagaaat aatgaagaac gaatggttcc gaaagaatct tcctgctgac
ttggtgaatg 960aaaatataat ggataaccaa tttgaagagc cagatcagcc
tatgcagagt atggatacga 1020tcatgcagat aatttcagaa gctaccgtac
cagcagctgg gagctattat tttgacgagt 1080ttatcgaagt ggatgaagat
atggatgaaa tagactctga ctatgaactt gatgtagata 1140gcagtggtga
gattgtatat gccatataat ttaatcatca tagaggtcac atattgaaaa
1200ggaagcacct tatattgagc tttatggctt tctcagcctc aaagctaaaa
aaataaatat 1260tctgagacta ttttctgcag actggatgat gcacgaagtt
catcatgttg atttatatat 1320tgtatgcttt cttggaacat gcattgtcca
caccatttat aagtatcact tttgtgagtt 1380gaggcaacat gttttcgaat
ttgtagggat cttctttatt ccttaaaaaa agttccacaa 1440cttcaattta
ggatgtatat tggcataatt ttagaacgtg gcatggcata attgagattt
1500tatatgcatg aaatatggta acgagctctt gatttctttt caaaaaaaaa
aaaaaaaaa 15592349PRTVicia faba 2Met Asp Met Pro Pro Pro Ile Met
His Asp Ser Asp Arg Tyr Asp Phe 1 5 10 15Val Arg Asp Ile Gly Ser
Gly Asn Phe Gly Val Ala Arg Leu Met Thr 20 25 30Asp Lys Leu Thr Lys
Asp Leu Val Ala Val Lys Tyr Ile Glu Arg Gly 35 40 45Asp Lys Ile Asp
Glu Asn Val Lys Arg Glu Ile Ile Asn His Arg Ser 50 55 60Leu Arg His
Pro Asn Ile Val Arg Phe Lys Glu Val Ile Leu Thr Pro65 70 75 80Thr
His Leu Ala Ile Val Met Glu Tyr Ala Ser Gly Gly Glu Met Ser 85 90
95Asp Arg Ile Ser Lys Ala Gly Arg Phe Thr Glu Asp Glu Ala Arg Phe
100 105 110Phe Phe Gln Gln Leu Ile Ser Gly Val Ser Tyr Cys His Ser
Met Gln 115 120 125Val Cys His Arg Asp Leu Lys Leu Glu Asn Thr Leu
Leu Asp Gly Asp 130 135 140Pro Ala Leu His Leu Lys Ile Cys Asp Phe
Gly Tyr Ser Lys Ser Ser145 150 155 160Val Leu His Ser Gln Pro Lys
Ser Thr Val Gly Thr Pro Ala Tyr Ile 165 170 175Ala Pro Glu Val Leu
Leu Lys Gln Glu Tyr Asp Gly Lys Ile Ala Asp 180 185 190Val Trp Ser
Cys Gly Val Thr Leu Tyr Val Met Leu Val Gly Ser Tyr 195 200 205Pro
Phe Glu Asp Pro Asp Asn Pro Lys Asp Phe Arg Lys Thr Ile Gln 210 215
220Arg Val Leu Ser Val Gln Tyr Ser Val Pro Asp Phe Val Gln Ile
Ser225 230 235 240Pro Glu Cys Arg Asp Ile Ile Ser Arg Ile Phe Val
Phe Asp Pro Ala 245 250 255Glu Arg Ile Thr Ile Pro Glu Ile Met Lys
Asn Glu Trp Phe Arg Lys 260 265 270Asn Leu Pro Ala Asp Leu Val Asn
Glu Asn Ile Met Asp Asn Gln Phe 275 280 285Glu Glu Pro Asp Gln Pro
Met Gln Ser Met Asp Thr Ile Met Gln Ile 290 295 300Ile Ser Glu Ala
Thr Val Pro Ala Ala Gly Ser Tyr Tyr Phe Asp Glu305 310 315 320Phe
Ile Glu Val Asp Glu Asp Met Asp Glu Ile Asp Ser Asp Tyr Glu 325 330
335Leu Asp Val Asp Ser Ser Gly Glu Ile Val Tyr Ala Ile 340
34531454DNAArabidopsis thaliana 3tttttttttt tttttccatt tattttctcg
aatcttcttc ttcttcctag attccagcga 60cttaacaaca acaacaacaa catattctct
gctgggtatt agattcgaat ttctcttttt 120gtgatcagaa atggatcgag
ctccggtgac cacaggaccg ttggatatgc cgattatgca 180cgacagtgat
cgatatgact tcgttaagga tattggttct ggtaatttcg gtgttgctcg
240tcttatgaga gataaactca ctaaagagct tgttgctgtc aagtacatcg
agagaggaga 300caagattgat gaaaatgttc aaagggagat cattaaccac
aggtcactaa ggcatcctaa 360tattgtcaga tttaaagagg tcattttgac
gccgactcat ctggctatca taatggaata 420tgcttctggc ggtgaacttt
acgagcggat ttgcaatgca ggacggttta gtgaagatga 480ggctcggttc
ttctttcagc agcttctatc tggagtcagt tattgtcatt cgatgcaaat
540ttgccatcgt gacctgaagc tagagaatac attgttggat ggaagtcctg
ctcctcgatt 600aaaaatttgt gattttggat attcaaagtc ttctgttctt
cattcacaac caaagtcaac 660tgttggtact cctgcataca tcgctccaga
ggtactgctt cgtcaggaat atgatggcaa 720gattgcagat gtatggtcat
gtggtgtgac cttatacgtc atgttggttg gagcgtatcc 780gttcgaagat
ccagaagagc caagagacta tcggaaaaca atacagagaa tccttagcgt
840taaatactca atccctgatg acatacggat atcacctgaa tgctgtcatc
ttatttcaag 900aatcttcgtg gctgatcccg ctaccagaat aagcatacca
gagatcaaaa cccatagttg 960gttcttgaag aatctccctg ctgatctaat
gaacgagagc aacacaggaa gccagttcca 1020ggagcctgaa caaccaatgc
aaagccttga cacaatcatg caaatcatct ctgaagccac 1080aattcccgct
gttcgaaacc gttgcctaga cgatttcatg actgacaatc ttgatcttga
1140cgatgacatg gatgactttg actctgaatc tgaaatcgac attgacagta
gcggagagat 1200agtttacgct ctctaataaa aagccttttt taacaaccaa
aacacttctc tatctgttct 1260aagaccagta gtgttctgat cctctggttt
caaattctac caatttttgt attgtctctg 1320tttgtttctt gttttcttca
tgcacacata tatcatatat gtaatgtaaa atatcatctg 1380tgtatattat
atatatattc caatgtcaca caaaagcaaa ttaacagtta aaacagttga
1440agcaagttga ggtt 14544361PRTArabidopsis thaliana 4Met Asp Arg
Ala Pro Val Thr Thr Gly Pro Leu Asp Met Pro Ile Met 1 5 10 15His
Asp Ser Asp Arg Tyr Asp Phe Val Lys Asp Ile Gly Ser Gly Asn 20 25
30Phe Gly Val Ala Arg Leu Met Arg Asp Lys Leu Thr Lys Glu Leu Val
35 40 45Ala Val Lys Tyr Ile Glu Arg Gly Asp Lys Ile Asp Glu Asn Val
Gln 50 55 60Arg Glu Ile Ile Asn His Arg Ser Leu Arg His Pro Asn Ile
Val Arg65 70 75 80Phe Lys Glu Val Ile Leu Thr Pro Thr His Leu Ala
Ile Ile Met Glu 85 90 95Tyr Ala Ser Gly Gly Glu Leu Tyr Glu Arg Ile
Cys Asn Ala Gly Arg 100 105 110Phe Ser Glu Asp Glu Ala Arg Phe Phe
Phe Gln Gln Leu Leu Ser Gly 115 120 125Val Ser Tyr Cys His Ser Met
Gln Ile Cys His Arg Asp Leu Lys Leu 130 135 140Glu Asn Thr Leu Leu
Asp Gly Ser Pro Ala Pro Arg Leu Lys Ile Cys145 150 155 160Asp Phe
Gly Tyr Ser Lys Ser Ser Val Leu His Ser Gln Pro Lys Ser 165 170
175Thr Val Gly Thr Pro Ala Tyr Ile Ala Pro Glu Val Leu Leu Arg Gln
180 185 190Glu Tyr Asp Gly Lys Ile Ala Asp Val Trp Ser Cys Gly Val
Thr Leu 195 200 205Tyr Val Met Leu Val Gly Ala Tyr Pro Phe Glu Asp
Pro Glu Glu Pro 210 215 220Arg Asp Tyr Arg Lys Thr Ile Gln Arg Ile
Leu Ser Val Lys Tyr Ser225 230 235 240Ile Pro Asp Asp Ile Arg Ile
Ser Pro Glu Cys Cys His Leu Ile Ser 245 250 255Arg Ile Phe Val Ala
Asp Pro Ala Thr Arg Ile Ser Ile Pro Glu Ile 260 265 270Lys Thr His
Ser Trp Phe Leu Lys Asn Leu Pro Ala Asp Leu Met Asn 275 280 285Glu
Ser Asn Thr Gly Ser Gln Phe Gln Glu Pro Glu Gln Pro Met Gln 290 295
300Ser Leu Asp Thr Ile Met Gln Ile Ile Ser Glu Ala Thr Ile Pro
Ala305 310 315 320Val Arg Asn Arg Cys Leu Asp Asp Phe Met Thr Asp
Asn Leu Asp Leu 325 330 335Asp Asp Asp Met Asp Asp Phe Asp Ser Glu
Ser Glu Ile Asp Ile Asp 340 345 350Ser Ser Gly Glu Ile Val Tyr Ala
Leu 355 3605357PRTArabidopsis thaliana 5Met Asp Arg Pro Ala Val Ser
Gly Pro Met Asp Leu Pro Ile Met His 1 5 10 15Asp Ser Asp Arg Tyr
Glu Leu Val Lys Asp Ile Gly Ser Gly Asn Phe 20 25 30Gly Val Ala Arg
Leu Met Arg Asp Lys Gln Ser Asn Glu Leu Val Ala 35 40 45Val Lys Tyr
Ile Glu Arg Gly Glu Lys Ile Asp Glu Asn Val Lys Arg 50 55 60Glu Ile
Ile Asn His Arg Ser Leu Arg His Pro Asn Ile Val Arg Phe65 70 75
80Lys Glu Val Ile Leu Thr Pro Thr His Leu Ala Ile Val Met Glu Tyr
85 90 95Ala Ser Gly Gly Glu Leu Phe Glu Arg Ile Cys Asn Ala Gly Arg
Phe 100 105 110Ser Glu Asp Glu Ala Arg Phe Phe Phe Gln Gln Leu Ile
Ser Gly Val 115 120 125Ser Tyr Cys His Ala Met Gln Val Cys His Arg
Asp Leu Lys Leu Glu 130 135 140Asn Thr Leu Leu Asp Gly Ser Pro Ala
Pro Arg Leu Lys Ile Cys Asp145 150 155 160Phe Gly Tyr Ser Lys Ser
Ser Val Leu His Ser Gln Pro Lys Ser Thr 165 170 175Val Gly Thr Pro
Ala Tyr Ile Ala Pro Glu Val Leu Leu Lys Lys Glu 180 185 190Tyr Asp
Gly Lys Val Ala Asp Val Trp Ser Cys Gly Val Thr Leu Tyr 195 200
205Val Met Leu Val Gly Ala Tyr Pro Phe Glu Asp Pro Glu Glu Pro Lys
210 215 220Asn Phe Arg Lys Thr Ile His Arg Ile Leu Asn Val Gln Tyr
Ala Ile225 230 235 240Pro Asp Tyr Val His Ile Ser Pro Glu Cys Arg
His Leu Ile Ser Arg 245 250 255Ile Phe Val Ala Asp Pro Ala Lys Arg
Ile Ser Ile Pro Glu Ile Arg 260 265 270Asn His Glu Trp Phe Leu Lys
Asn Leu Pro Ala Asp Leu Met Asn Asp 275 280 285Asn Thr Met Thr Thr
Gln Phe Asp Glu Ser Asp Gln Pro Gly Gln Ser 290 295 300Ile Glu Glu
Ile Met Gln Ile Ile Ala Glu Ala Thr Val Pro Pro Ala305 310 315
320Gly Thr Gln Asn Leu Asn His Tyr Leu Thr Asp Asp Asp Met Glu Glu
325 330 335Asp Leu Glu Ser Asp Leu Asp Asp Leu Asp Ile Asp Ser Ser
Gly Glu 340 345 350Ile Val Tyr Ala Met 35561448DNAArabidopsis
thaliana 6ggaattccct ttttccccca aattcatatc cttccttaga tatttttctc
cttcttcttc 60ttctagattc cagctactcc agaagattct tcgacttaat ctgatgtgat
taggaagagc 120aatagaggaa gagaatcaga aaaaatggat ccggcgacta
attcaccgat tatgccgatt 180gatttaccga ttatgcacga cagtgatcgt
tacgacttcg ttaaagatat tggctctggt 240aatttcggcg ttgctcgtct
catgaccgat agagtcacca aggagcttgt tgctgttaaa 300tacatcgaga
gaggagaaaa gattgatgaa aatgttcaga gggagattat caatcataga
360tcattgagac atcctaatat tgttaggttt aaagaggtga ttttgacgcc
ttcccatttg 420gctattgtta tggaatatgc tgctggtgga gaactttatg
agcggatttg taatgccgga 480cggtttagtg aagatgaggc tcggttcttc
tttcagcagc ttatatctgg agttagctat 540tgtcatgcaa tgcaaatatg
ccatcgggat ctgaagctgg aaaatacatt gttagatgga 600agtccggcac
ctcgtttgaa aatatgtgat tttggttatt ccaagtcttc tgttcttcat
660tcccaaccaa agtcaactgt tggtactcct gcatacattg caccagagat
tcttcttcga 720caggaatatg atggcaagct tgcagatgta tggtcttgcg
gtgtaacatt atatgtaatg 780ttggttggag cttatccatt cgaggatcca
caggagccac gagattatcg aaagacaata 840caaagaatcc ttagtgtcac
atactcgatc ccagaggact tacacctctc accagaatgt 900cgccatctaa
tatcgaggat cttcgtggct gatccggcaa caagaatcac tattccggag
960atcacatccg ataaatggtt cttgaagaat ctaccaggtg atttgatgga
tgagaaccga 1020atgggaagtc agtttcaaga gcctgagcag ccaatgcaga
gccttgacac gattatgcag 1080ataatatcgg aggctacgat tccgactgtt
cgtaatcgtt gcctcgatga tttcatggcg 1140gataatcttg atctagacga
tgacatggat gactttgatt ccgaatctga gattgatgtt 1200gacagtagtg
gagagatagt ttatgctctc tgagattcct gaggacaaag tctgttttgt
1260ccgtactgtt gagacacacc actggagttt tgtcttagct ccacgcactc
catcgttcat 1320ttttggatcg tttgttgttt tttactctac aagctttgga
ttcacataca tatatatgta 1380ttgtaatgta atatgtaata tattctatgt
atttctcttt gtttaataac tattggcaca 1440ttttatac
14487362PRTArabidopsis thaliana 7Met Asp Pro Ala Thr Asn Ser Pro
Ile Met Pro Ile Asp Leu Pro Ile 1 5 10 15Met His Asp Ser Asp Arg
Tyr Asp Phe Val Lys Asp Ile Gly Ser Gly 20 25 30Asn Phe Gly Val Ala
Arg Leu Met Thr Asp Arg Val Thr Lys Glu Leu 35 40 45Val Ala Val Lys
Tyr Ile Glu Arg Gly Glu Lys Ile Asp Glu Asn Val 50 55 60Gln Arg Glu
Ile Ile Asn His Arg Ser Leu Arg His Pro Asn Ile Val65 70 75 80Arg
Phe Lys Glu Val Ile Leu Thr Pro Ser His Leu Ala Ile Val Met 85 90
95Glu Tyr Ala Ala Gly Gly Glu Leu Tyr Glu Arg Ile Cys Asn Ala Gly
100 105 110Arg Phe Ser Glu Asp Glu Ala Arg Phe Phe Phe Gln Gln Leu
Ile Ser 115 120 125Gly Val Ser Tyr Cys His Ala Met Gln Ile Cys His
Arg Asp Leu Lys 130 135 140Leu Glu Asn Thr Leu Leu Asp Gly Ser Pro
Ala Pro Arg Leu Lys Ile145 150 155 160Cys Asp Phe Gly Tyr Ser Lys
Ser Ser Val Leu His Ser Gln Pro Lys 165 170 175Ser Thr Val Gly Thr
Pro Ala Tyr Ile Ala Pro Glu Ile Leu Leu Arg 180 185 190Gln Glu Tyr
Asp Gly Lys Leu Ala Asp Val Trp Ser Cys Gly Val Thr 195 200 205Leu
Tyr Val Met Leu Val Gly Ala Tyr Pro Phe Glu Asp Pro Gln Glu 210 215
220Pro Arg Asp Tyr Arg Lys Thr Ile Gln Arg Ile Leu Ser Val Thr
Tyr225 230 235 240Ser Ile Pro Glu Asp Leu His Leu Ser Pro Glu Cys
Arg His Leu Ile 245 250 255Ser Arg Ile Phe Val Ala Asp Pro Ala Thr
Arg Ile Thr Ile Pro Glu 260 265 270Ile Thr Ser Asp Lys Trp Phe Leu
Lys Asn Leu Pro Gly Asp Leu Met 275 280 285Asp Glu Asn Arg Met Gly
Ser Gln Phe Gln Glu Pro Glu Gln Pro Met 290 295 300Gln Ser Leu Asp
Thr Ile Met Gln Ile Ile Ser Glu Ala Thr Ile Pro305 310 315 320Thr
Val Arg Asn Arg Cys Leu Asp Asp Phe Met Ala Asp Asn Leu Asp 325 330
335Leu Asp Asp Asp Met Asp Asp Phe Asp Ser Glu Ser Glu Ile Asp Val
340 345 350Asp Ser Ser Gly Glu Ile Val Tyr Ala Leu 355
3608362PRTArabidopsis thaliana 8Met Asp Pro Ala Thr Asn Ser Pro Ile
Met Pro Ile Asp Leu Pro Ile 1 5 10 15Met His Asp Ser Asp Arg Tyr
Asp Phe Val Lys Asp Ile Gly Ser Gly 20 25 30Asn Phe Gly Val Ala Arg
Leu Met Thr Asp Arg Val Thr Lys Glu Leu 35 40 45Val Ala Val Lys Tyr
Ile Glu Arg Gly Glu Lys Ile Asp Glu Asn Val 50 55 60Gln Arg Glu Ile
Ile Asn His Arg Ser Leu Arg His Pro Asn Ile Val65 70 75 80Arg Phe
Lys Glu Val Ile Leu Thr Pro Ser His Leu Ala Ile Val Met 85 90 95Glu
Tyr Ala Ala Gly Gly Glu Leu Tyr Glu Arg Ile Cys Asn Ala Gly 100 105
110Arg Phe Ser Glu Asp Glu Ala Arg Phe Phe Phe Gln Gln Leu Ile Ser
115 120 125Gly Val Ser Tyr Cys His Ala Met Gln Ile Cys His Arg Asp
Leu Lys 130 135 140Leu Glu Asn Thr Leu Leu Asp Gly Ser Pro Ala Pro
Arg Leu Lys Ile145 150 155 160Cys Asp Phe Gly Tyr Ser Lys Ser Ser
Val Leu His Ser Gln Pro Lys 165 170 175Ser Thr Val Gly Thr Pro Ala
Tyr Ile Ala Pro Glu Ile Leu Leu Arg 180 185 190Gln Glu Tyr Asp Gly
Lys Leu Ala Asp Val Trp Ser Cys Gly Val Thr 195 200 205Leu Tyr Val
Met Leu Val Gly Ala Tyr Pro Phe Glu Asp Pro Gln Glu 210 215 220Pro
Arg Asp Tyr Arg Lys Thr Ile Gln Arg Ile Leu Ser Val Thr Tyr225 230
235 240Ser Ile Pro Glu Asp Leu His Leu Ser Pro Glu Cys Arg His Leu
Ile 245 250 255Ser Arg Ile Phe Val Ala Asp Pro Ala Thr Arg Ile Thr
Ile Pro Glu 260
265 270Ile Thr Ser Asp Lys Trp Phe Leu Lys Asn Leu Pro Gly Asp Leu
Met 275 280 285Asp Glu Asn Arg Met Gly Ser Gln Phe Gln Glu Pro Glu
Gln Pro Met 290 295 300Gln Ser Leu Asp Thr Ile Met Gln Ile Ile Ser
Glu Ala Thr Ile Pro305 310 315 320Thr Val Arg Asn Arg Cys Leu Asp
Asp Phe Met Ala Asp Asn Leu Asp 325 330 335Leu Asp Asp Asp Met Asp
Asp Phe Asp Ser Glu Ser Glu Ile Asp Val 340 345 350Asp Ser Ser Gly
Glu Ile Val Tyr Ala Leu 355 36091210DNATriticum aestivum
9ggttccggca acttcggggt ggccaagctg gtgcgggacg tccggaccaa ggagcacttc
60gccgtcaagt tcatcgagcg aggccacaag attgatgaac atgttcaaag ggagattatg
120aaccaccggt cactcaagca tccaaatatt attcgattca aggaggtcgt
gctaactccc 180acacatttgg caatagttat ggaatatgcc tctggcggcg
agctatttca aaggatttgc 240aacgcaggga gatttagcga ggatgaggga
agattcttct tccaacaatt gatttctgga 300gtgagctatt gtcactctat
gcaagtatgt catagagatt tgaaactaga aaacactctc 360ttggatggta
gtgtcgcacc tcggctcaag atttgtgact tcggttactc caagtcttct
420gtcttgcact ctcaaccgaa gtcaactgtc ggcacaccgg catacatcgc
cccagaggtc 480ctctctagaa gagaatatga tggaaaggtc gccgatgttt
ggtcatgcgg agtaacgctc 540tatgtgatgc ttgtcggggc atatcctttc
gaggaccctg atgagccaag gaacttccgc 600aaaacgatca ctaggatact
cagcgtacag tactctgttc cggactacgt tcgagtctcg 660atggactgca
tacatctact gtcccgcatt ttcgttggaa atcctcagca gcgaataacc
720atcccggaga tcaagaacca tccatggttc ctcaagagat tgcccgttga
gatgaccgat 780gagtaccaaa ggagcatgca gctggcagac atgaacacgc
cgtcacagag cctggaagaa 840gccatggcga tcatccagga ggcgcagaaa
cctggcgata acgccctagg ggttgccggg 900caggttgcct gcctggggag
catggatttg gacgacatcg atttcgatat cgacgacatt 960gacgttgaga
gcagcgggga tttcgtgtgc ccgttgtgat tgctcatgag tggttcaaaa
1020gttctcttga tggtttgcct gtggatggat ccctgttttg tcatgcttcc
actagatttt 1080gttctgggtc acaaattctc tgtagcctac agattggctt
gatgtgtaaa cagtgtaaga 1140taagtttaca tgcttatatc gaaatcagta
gttttacccg aaaaaaaaaa aaaaaaaaaa 1200aaaaaaaaaa
121010332PRTTriticum aestivum 10Gly Ser Gly Asn Phe Gly Val Ala Lys
Leu Val Arg Asp Val Arg Thr 1 5 10 15Lys Glu His Phe Ala Val Lys
Phe Ile Glu Arg Gly His Lys Ile Asp 20 25 30Glu His Val Gln Arg Glu
Ile Met Asn His Arg Ser Leu Lys His Pro 35 40 45Asn Ile Ile Arg Phe
Lys Glu Val Val Leu Thr Pro Thr His Leu Ala 50 55 60Ile Val Met Glu
Tyr Ala Ser Gly Gly Glu Leu Phe Gln Arg Ile Cys65 70 75 80Asn Ala
Gly Arg Phe Ser Glu Asp Glu Gly Arg Phe Phe Phe Gln Gln 85 90 95Leu
Ile Ser Gly Val Ser Tyr Cys His Ser Met Gln Val Cys His Arg 100 105
110Asp Leu Lys Leu Glu Asn Thr Leu Leu Asp Gly Ser Val Ala Pro Arg
115 120 125Leu Lys Ile Cys Asp Phe Gly Tyr Ser Lys Ser Ser Val Leu
His Ser 130 135 140Gln Pro Lys Ser Thr Val Gly Thr Pro Ala Tyr Ile
Ala Pro Glu Val145 150 155 160Leu Ser Arg Arg Glu Tyr Asp Gly Lys
Val Ala Asp Val Trp Ser Cys 165 170 175Gly Val Thr Leu Tyr Val Met
Leu Val Gly Ala Tyr Pro Phe Glu Asp 180 185 190Pro Asp Glu Pro Arg
Asn Phe Arg Lys Thr Ile Thr Arg Ile Leu Ser 195 200 205Val Gln Tyr
Ser Val Pro Asp Tyr Val Arg Val Ser Met Asp Cys Ile 210 215 220His
Leu Leu Ser Arg Ile Phe Val Gly Asn Pro Gln Gln Arg Ile Thr225 230
235 240Ile Pro Glu Ile Lys Asn His Pro Trp Phe Leu Lys Arg Leu Pro
Val 245 250 255Glu Met Thr Asp Glu Tyr Gln Arg Ser Met Gln Leu Ala
Asp Met Asn 260 265 270Thr Pro Ser Gln Ser Leu Glu Glu Ala Met Ala
Ile Ile Gln Glu Ala 275 280 285Gln Lys Pro Gly Asp Asn Ala Leu Gly
Val Ala Gly Gln Val Ala Cys 290 295 300Leu Gly Ser Met Asp Leu Asp
Asp Ile Asp Phe Asp Ile Asp Asp Ile305 310 315 320Asp Val Glu Ser
Ser Gly Asp Phe Val Cys Pro Leu 325 33011339PRTNicotiana tabacum
11Met Glu Glu Lys Tyr Glu Leu Leu Lys Glu Leu Gly Thr Gly Asn Phe 1
5 10 15Gly Val Ala Arg Leu Val Lys Asp Lys Lys Thr Lys Glu Leu Phe
Ala 20 25 30Val Lys Tyr Ile Glu Arg Gly Lys Lys Ile Asp Glu Asn Val
Gln Arg 35 40 45Glu Ile Ile Asn His Arg Ser Leu Gly His Pro Asn Ile
Ile Arg Phe 50 55 60Lys Glu Val Leu Val Thr Pro Ser His Leu Ala Ile
Val Met Glu Tyr65 70 75 80Ala Ala Gly Gly Glu Leu Phe Ala Arg Ile
Cys Ser Ala Gly Arg Phe 85 90 95Ser Glu Asp Glu Ala Arg Phe Phe Phe
Gln Gln Leu Ile Ser Gly Val 100 105 110Ser Tyr Cys His Ala Met Glu
Ile Cys His Arg Asp Leu Lys Leu Glu 115 120 125Asn Thr Leu Leu Asp
Gly Ser Ala Ser Pro Arg Val Lys Ile Cys Asp 130 135 140Phe Gly Tyr
Ser Lys Ser Gly Leu Leu His Ser Gln Pro Lys Ser Thr145 150 155
160Val Gly Thr Pro Ala Tyr Ile Ala Pro Glu Val Leu Ser Arg Lys Glu
165 170 175Tyr Asp Gly Lys Ile Ala Asp Val Trp Ser Cys Gly Val Thr
Leu Tyr 180 185 190Val Met Leu Val Gly Ala Tyr Pro Phe Glu Asp Pro
Glu Asp Pro Lys 195 200 205Asn Phe Arg Lys Thr Ile Gly Arg Ile Met
Ser Ala Gln Tyr Ser Ile 210 215 220Pro Asp Tyr Val Arg Ile Ser Ala
Asp Cys Lys Asn Leu Leu Ser Arg225 230 235 240Ile Phe Val Ala Asn
Pro Ser Lys Arg Ile Thr Ile Pro Glu Ile Lys 245 250 255Lys His Pro
Trp Phe Leu Lys Asn Leu Pro Lys Asp Leu Met Asp Gly 260 265 270Glu
His Ser Lys Tyr Glu Glu Ala Ser Glu Gln Leu Gln Gln Ser Val 275 280
285Glu Glu Ile Met Arg Ile Ile Gln Glu Ala Lys Ile Pro Gly Glu Val
290 295 300Ser Lys Pro Glu Gly Gln Ala Thr Ala Gly Thr Ala Glu Pro
Asp Asp305 310 315 320Thr Glu Asp Asp Leu Glu Ser Glu Ile Asp Ser
Ser Asn Asp Phe Ala 325 330 335Val Tyr Val12349PRTGlycine max 12Met
Asp Lys Tyr Glu Ala Val Lys Asp Leu Gly Ala Gly Asn Phe Gly 1 5 10
15Val Ala Arg Leu Met Arg Asn Lys Val Thr Lys Glu Leu Val Ala Met
20 25 30Lys Tyr Ile Glu Arg Gly Pro Lys Ile Asp Glu Asn Val Ala Arg
Glu 35 40 45Ile Met Asn His Arg Ser Leu Arg His Pro Asn Ile Ile Arg
Tyr Lys 50 55 60Glu Val Val Leu Thr Pro Thr His Leu Ala Ile Val Met
Glu Tyr Ala65 70 75 80Ala Gly Gly Glu Leu Phe Glu Arg Ile Cys Ser
Ala Gly Arg Phe Ser 85 90 95Glu Asp Glu Ala Arg Tyr Phe Phe Gln Gln
Leu Ile Ser Gly Val His 100 105 110Phe Cys His Thr Met Gln Ile Cys
His Arg Asp Leu Lys Leu Glu Asn 115 120 125Thr Leu Leu Asp Gly Ser
Pro Ala Pro Arg Leu Lys Ile Cys Asp Phe 130 135 140Gly Tyr Ser Lys
Ser Ser Leu Leu His Ser Arg Pro Lys Ser Thr Val145 150 155 160Gly
Thr Pro Ala Tyr Ile Ala Pro Glu Val Leu Ser Arg Arg Glu Tyr 165 170
175Asp Gly Lys Leu Ala Asp Val Trp Ser Cys Ala Val Thr Leu Tyr Val
180 185 190Met Leu Val Gly Ala Tyr Pro Phe Glu Asp Gln Asp Asp Pro
Arg Asn 195 200 205Phe Arg Lys Thr Ile Gln Arg Ile Met Ala Val Gln
Tyr Lys Ile Pro 210 215 220Asp Tyr Val His Ile Ser Gln Asp Cys Arg
His Leu Leu Ser Arg Ile225 230 235 240Phe Val Ala Asn Pro Leu Arg
Arg Ile Thr Ile Lys Glu Ile Lys Asn 245 250 255His Pro Trp Phe Leu
Arg Asn Leu Pro Arg Glu Leu Thr Glu Ser Ala 260 265 270Gln Ala Ile
Tyr Tyr Gln Arg Asp Ser Pro Asn Phe His Leu Gln Ser 275 280 285Val
Asp Glu Ile Met Lys Ile Val Gly Glu Ala Arg Asn Pro Pro Pro 290 295
300Val Ser Arg Ala Leu Lys Gly Phe Gly Trp Glu Gly Glu Glu Asp
Leu305 310 315 320Asp Glu Glu Val Glu Glu Glu Glu Asp Glu Asp Glu
Tyr Asp Lys Arg 325 330 335Val Lys Glu Val His Ala Ser Gly Glu Phe
Gln Ile Ser 340 34513334PRTOryza sativa 13Met Glu Glu Arg Tyr Glu
Ala Leu Lys Glu Leu Gly Ala Gly Asn Phe 1 5 10 15Gly Val Ala Arg
Leu Val Arg Asp Lys Arg Ser Lys Glu Leu Val Ala 20 25 30Val Lys Tyr
Ile Glu Arg Gly Lys Lys Ile Asp Glu Asn Val Gln Arg 35 40 45Glu Ile
Ile Asn His Arg Ser Leu Arg His Pro Asn Ile Ile Arg Phe 50 55 60Lys
Glu Val Cys Leu Thr Pro Thr His Leu Ala Ile Val Met Glu Tyr65 70 75
80Ala Ala Gly Gly Glu Leu Phe Glu Gln Ile Cys Thr Ala Gly Arg Phe
85 90 95Ser Glu Asp Asp Ala Arg Tyr Phe Phe Gln Gln Leu Ile Ser Gly
Val 100 105 110Ser Tyr Cys His Ser Leu Glu Ile Cys His Arg Asp Leu
Lys Leu Glu 115 120 125Asn Thr Leu Leu Asp Gly Ser Pro Thr Pro Arg
Val Lys Ile Cys Asp 130 135 140Phe Gly Tyr Ser Lys Ser Ala Leu Leu
His Ser Lys Pro Lys Ser Thr145 150 155 160Val Gly Thr Pro Ala Tyr
Ile Ala Pro Glu Val Leu Ser Arg Lys Glu 165 170 175Tyr Asp Gly Lys
Val Ala Asp Val Trp Ser Cys Gly Val Thr Leu Tyr 180 185 190Val Met
Leu Val Gly Ser Tyr Pro Phe Glu Asp Pro Gly Asp Pro Arg 195 200
205Asn Phe Arg Lys Thr Ile Ser Arg Ile Leu Gly Val Gln Tyr Ser Ile
210 215 220Pro Asp Tyr Val Arg Val Ser Ser Asp Cys Arg Arg Leu Leu
Ser Gln225 230 235 240Ile Phe Val Ala Asp Pro Ser Lys Arg Ile Thr
Ile Pro Glu Ile Lys 245 250 255Lys His Thr Trp Phe Leu Lys Asn Leu
Pro Lys Glu Ile Ser Glu Arg 260 265 270Glu Lys Ala Asp Tyr Lys Asp
Thr Asp Ala Ala Pro Pro Thr Gln Ala 275 280 285Val Glu Glu Ile Met
Arg Ile Ile Gln Glu Gly Lys Val Pro Gly Asp 290 295 300Met Ala Ala
Ala Asp Pro Ala Leu Leu Ala Glu Leu Ala Glu Leu Lys305 310 315
320Ser Asp Asp Glu Glu Glu Ala Ala Asp Glu Tyr Asp Thr Tyr 325
33014342PRTMesembryanthemum crystallinum 14Met Glu Leu Tyr Glu Ile
Val Lys Asp Ile Gly Ser Gly Asn Phe Gly 1 5 10 15Gln Ala Lys Leu
Val Arg Asp Lys Trp Thr Asn Glu Phe Val Ala Val 20 25 30Lys Phe Ile
Glu Arg Gly Ser Lys Asp Asp Glu His Val Gln Arg Lys 35 40 45Leu Met
Asn His Ser Ser Leu Lys His Pro Asn Ile Ile Arg Phe Lys 50 55 60Glu
Val Leu Leu Thr Pro Thr His Leu Ala Ile Val Met Glu Tyr Ala65 70 75
80Ala Gly Gly Glu Leu Phe Glu Arg Ile Cys Asn Ala Gly Arg Phe Arg
85 90 95Glu Asp Glu Ala Arg Phe Phe Phe Gln Gln Leu Ile Ser Gly Val
Ser 100 105 110Tyr Cys His Ser Met Gln Ile Cys His Arg Asp Leu Lys
Leu Glu Asn 115 120 125Thr Leu Leu Asp Gly Ser Pro Ala Pro Arg Val
Lys Ile Cys Asp Phe 130 135 140Gly Tyr Ser Lys Ser Ser Val Leu His
Ser Gln Pro Lys Ser Ala Val145 150 155 160Gly Thr Pro Ala Tyr Ile
Ala Pro Glu Val Leu Ser Lys Arg Glu Tyr 165 170 175Asp Gly Lys Ile
Ala Asp Val Trp Ser Cys Gly Val Thr Leu Tyr Val 180 185 190Met Leu
Phe Gly Ala Tyr Pro Phe Glu Asp Pro Asp Asp Pro Lys Asn 195 200
205Phe Arg Lys Ser Leu Val Arg Ile Leu Ser Val Gln Tyr Cys Ile Pro
210 215 220Asp Asn Ile Pro Ile Ser Met Glu Cys Arg His Leu Leu Ser
Arg Ile225 230 235 240Phe Val Ala Asn Pro Glu Lys Arg Ile Thr Ile
Pro Glu Ile Lys Asn 245 250 255His Pro Trp Phe Gln Lys Asn Leu Pro
Met Glu Leu Met Glu Gly Gly 260 265 270Ser Trp Gln Ser His Asp Ile
Asn His Pro Ser Gln Asn Ile Gly Glu 275 280 285Ile Leu Ser Ile Ile
Gln Glu Ala Arg Gln Pro Ala Glu Leu Pro Ser 290 295 300Thr Gly Gly
Leu Gln Ile Gly Gly Thr Leu Asp Phe Asp Asp Leu Asp305 310 315
320Val Asp Leu Asp Val Asp Val Asp Leu Asp Asp Ile Glu Ser Ser Gly
325 330 335Glu Phe Val Cys Pro Met 3401511PRTArtificial
SequenceSynthetic Sequence 15Pro Ile Met His Asp Ser Asp Arg Tyr
Asp Phe 1 5 101619PRTArtificial SequenceSynthetic Sequence 16Pro
Ala Asp Leu Val Asn Glu Asn Ile Met Asp Asn Gln Phe Glu Glu 1 5 10
15Pro Asp Gln1720DNAArtificial SequenceSynthetic Sequence
17ttgcyrtyaa rtacatcgaa 201820DNAArtificial Sequence 18ccatcyarna
gngtrttttc 201930DNAArtificial Sequence 19gaatctccac tacgacgccg
tttacttccc 302030DNAArtificial Sequence 20ccgtgcaacc atggatatgg
catatacaat 30
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