U.S. patent application number 12/723779 was filed with the patent office on 2010-07-08 for rubisco activase with increased thermostability and methods of use thereof.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to Itzhak Kurek, Lu Liu, Genhai Zhu.
Application Number | 20100175151 12/723779 |
Document ID | / |
Family ID | 37726793 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100175151 |
Kind Code |
A1 |
Kurek; Itzhak ; et
al. |
July 8, 2010 |
RUBISCO ACTIVASE WITH INCREASED THERMOSTABILITY AND METHODS OF USE
THEREOF
Abstract
The present invention provides thermostable polypeptides related
to Arabidopsis Rubisco Activase polypeptides. Nucleic acids
encoding the polypeptides of the invention are also provided.
Methods for using the polypeptides and nuclei acids of the
invention to enhance resistance of plants to heat stress are
encompassed.
Inventors: |
Kurek; Itzhak; (San
Francisco, CA) ; Liu; Lu; (Palo Alto, CA) ;
Zhu; Genhai; (San Jose, CA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL, INC.
7250 N.W. 62ND AVENUE, P.O. BOX 552
JOHNSTON
IA
50131-0552
US
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
Johnston
IA
|
Family ID: |
37726793 |
Appl. No.: |
12/723779 |
Filed: |
March 15, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12474298 |
May 29, 2009 |
7723573 |
|
|
12723779 |
|
|
|
|
11867723 |
Oct 5, 2007 |
7557267 |
|
|
12474298 |
|
|
|
|
11507729 |
Aug 22, 2006 |
7314975 |
|
|
11867723 |
|
|
|
|
60711449 |
Aug 24, 2005 |
|
|
|
60733110 |
Nov 2, 2005 |
|
|
|
Current U.S.
Class: |
800/289 ;
435/252.31; 435/252.33; 435/254.2; 435/320.1; 435/348; 435/419;
530/350; 536/23.1; 800/298; 800/305; 800/312; 800/314; 800/317.2;
800/317.3; 800/317.4; 800/320.1; 800/320.2; 800/322 |
Current CPC
Class: |
C12N 9/88 20130101; C12N
15/8261 20130101; Y02A 40/146 20180101 |
Class at
Publication: |
800/289 ;
536/23.1; 435/320.1; 530/350; 435/252.33; 435/252.31; 435/254.2;
435/419; 435/348; 800/298; 800/320.1; 800/317.4; 800/317.2;
800/320.2; 800/312; 800/314; 800/322; 800/305; 800/317.3 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 15/11 20060101 C12N015/11; C12N 15/63 20060101
C12N015/63; C07K 14/00 20060101 C07K014/00; C12N 1/21 20060101
C12N001/21; C12N 1/19 20060101 C12N001/19; C12N 5/10 20060101
C12N005/10; A01H 5/00 20060101 A01H005/00 |
Claims
1. An isolated nucleic acid molecule comprising any of SEQ ID NOS:
3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a complement thereof.
2. An isolated nucleic acid molecule selected from the group
consisting of: a. a nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to the nucleotide sequence
of any of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a
complement thereof; b. a nucleic acid molecule that encodes a
polypeptide comprising the amino acid sequence of any of SEQ ID
NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22; and c. a nucleic acid
molecule that hybridizes under stringent conditions with a nucleic
acid probe consisting of the nucleotide sequence of any of SEQ ID
NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a complement
thereof.
3. The isolated nucleic acid molecule of claim 1 or 2, wherein the
nucleic acid molecule encodes a polypeptide with increased
thermostability as compared to a polypeptide of SEQ ID NO: 2.
4. A vector comprising a nucleic acid molecule of claim 1 or 2.
5. The vector of claim 4 that is an expression vector.
6. A host cell which comprises the vector of claim 4.
7. An isolated polypeptide comprising any one of SEQ ID NOS: 4, 6,
8, 10, 12, 14, 16, 18, 20, 22.
8. An isolated polypeptide selected from the group consisting of:
a. a polypeptide that is at least 95% identical to the amino acid
sequence of any of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22;
b. a polypeptide that is encoded by a nucleic acid molecule
comprising a nucleotide sequence that is at least 95% identical to
any one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a
complement thereof; c. polypeptide encoded by a nucleic acid
molecule that hybridizes under stringent conditions with a nucleic
acid probe consisting of the nucleotide sequence of any of SEQ ID
NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a complement
thereof.
9. The isolated polypeptide of claim 7 or 8, wherein the
polypeptide has increased thermostability as compared to a
polypeptide of SEQ ID NO: 2.
10. A transgenic plant comprising a transgene that expresses a. a
polypeptide of claim 8, or b. a nucleic acid molecule of claim
2.
11. The transgenic plant of claim 10, wherein the plant is selected
from the group consisting of maize, tomato, potato, rice, soybean,
cotton, sunflower, alfalfa, lettuce, canola, sorghum or tobacco
plants.
12. The transgenic plant of claim 10, wherein the transgenic plant
has increased heat tolerance as compared to a plant that is not
transgenic.
13. A method of increasing the heat tolerance in plants comprising
expressing a polypeptide of any of SEQ ID NOS: 4, 6, 8, 10, 12, 14,
16, 18, 20, 22 in the plant.
14. The method of claim 13, wherein the polypeptide is expressed in
the one or more plastids of the plant.
15. The method according to claim 13, wherein the expression of
said polypeptide results in an increase in photosynthesis rate
under heated conditions.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This utility application is a divisional application
claiming benefit from U.S. Utility patent application Ser. No.
12/474,298 filed May 29, 2009 which is a divisional of U.S. Utility
patent application Ser. No. 11/867,723, filed Oct. 5, 2007, now
granted U.S. Pat. No. 7,557,267 issued Jul. 7, 2009, which is a
divisional of U.S. Utility patent application Ser. No. 11/507,729,
filed Aug. 22, 2006, now granted U.S. Pat. No. 7,314,975 issued
Jan. 1, 2008, which also claims the benefit U.S. Provisional Patent
Application Ser. No. 60/711,449, filed Aug. 24, 2005 and U.S.
Provisional Serial Application No. 60/733,110, filed Nov. 2, 2005,
all of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to increasing the
levels of photosynthesis in plants grown under increased
temperatures. More particularly, the present invention relates to
improving the thermostability of the photosynthetic enzyme Rubisco
Activase.
BACKGROUND OF THE INVENTION
[0003] Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is
an important enzyme in the photosynthetic process. This enzyme
incorporates CO.sub.2 into plants during photosynthesis.
Atmospheric oxygen competes with CO.sub.2 as a substrate for
Rubisco, giving rise to photorespiration and making Rubisco the
rate-limiting step in photosynthesis.
[0004] Rubisco Activase (RCA) is a protein that catalyzes the
activation of Rubisco, which in turn, regulates photosynthesis by
initiating photosynthetic carbon reduction and photorespiratory
carbon oxidation. The Rubisco Activase enzyme catalyzes the release
of ribulose-1,5-bisphosphate (RuBP) from Rubisco. This newly
unoccupied site on Rubisco is now free to bind the CO.sub.2 and
Mg.sup.2+ activators in order for photosynthesis to proceed.
Rubisco Activase is also responsible for releasing sugar phosphate
inhibitors from Rubisco and restores Rubisco catalytic activity.
Thus, if Rubisco Activase is impaired, Rubisco remains inactive and
photosynthesis slows.
[0005] Rubisco Activase is thermo-labile and thus has decreasing
activity with increasing temperatures. As a result, the
photosynthetic process slows due to the lack of Rubisco activation.
The Rubisco Activase enzyme denatures under increased temperatures,
thus rendering the enzyme unable to convert inactive Rubisco to the
active form. Arabidopsis contains two RCA isoforms, the short
thermolabile (RCA1) and the long relatively thermostable (RCA2)
forms that are generated by alternative splicing of pre-mRNA
(Werneke, et al., (1989) Plant Cell 1:815-825).
[0006] Crop plants grown in hot climates could benefit from
increasing photosynthetic levels. Accordingly, if the rate limiting
step in photosynthesis could be made more heat tolerant, crop
plants could more easily grow in these climates.
SUMMARY OF THE INVENTION
[0007] The present invention relates to Rubisco Activase derived
polypeptides that are more thermostable than naturally occurring
Rubisco Activase. The Rubisco Activase derived polypeptides of the
invention substantially retain activity in plants grown under
conditions of increased temperature. Nucleic acid molecules
encoding the polypeptides of the invention are also
encompassed.
[0008] In addition to the Rubisco Activase derived polypeptides of
the invention, it will be appreciated that the invention also
encompasses variants thereof, including, but not limited to, any
substantially similar sequence, any fragment, analog, homolog,
mutant or modified polypeptide thereof. The variants encompassed by
the invention are at least partially functionally active (i.e.,
they are capable of displaying one or more known functional
activities associated with wild type Rubisco Activase) under heated
conditions. Nucleic acid molecules encoding the variant
polypeptides are also encompassed.
[0009] Vectors comprising one or more nucleic acids of the
invention are also encompassed.
[0010] Cells comprising a polypeptide, nucleic acid molecule and/or
vector of the invention are also encompassed.
[0011] The present invention also relates to transgenic plants
comprising a polypeptide, nucleic acid molecule, and/or vector of
the invention. The transgenic plants can express the transgene in
any way known in the art including, but not limited to,
constitutive expression, developmentally regulated expression,
tissue specific expression, etc. Seed obtained from a transgenic
plant of the invention is also encompassed.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIGS. 1A-1C: Characterization of wild type RCA (RCA1) and
thermostable variants (183H12, 301C7 and 382D8). (A) Rubisco
activity after activation by activase after treatment at 25.degree.
C. (white), 40.degree. C. (gray) and 45.degree. C. (black).
Activase proteins were incubated at the indicated temperatures for
15 min prior to assay at 25.degree. C. (B) Activation of Rubisco
under catalytic conditions at 25.degree. C. (white) and 40.degree.
C. (gray). (C) ATPase activity of activase proteins incubated at
the indicated temperatures for 15 min prior to assay at 25.degree.
C.
[0013] FIGS. 2A-2D: Characterization of the Rubisco Activase mutant
(.DELTA.rca) at ambient CO.sub.2. (A) Immuno blot analysis from
leaves of Arabidopsis wild-type (RCA/RCA), heterozygous
(RCA/.DELTA.rca) and homozygous (.DELTA.rca/.DELTA.rca) plants. The
blot was immunodecorated with polyclonal antibodies raised against
the recombinant Arabidopsis RCA1. (B) Photosynthetic performance of
three-week old wild-type (upper) and .DELTA.rca (lower) plants as
measured using fluorescence image analysis. (C) Leaf area of the
plants described in (B) (50 plants per phenotype) at the indicted
age. (D) Photograph of eight week old wild-type (upper) and
.DELTA.rca (lower) plants.
[0014] FIGS. 3A-3C: Molecular characterization of .DELTA.rca
mutant. (A) Schematic map of the wild-type (RCA) and deletion
(.DELTA.rca) alleles. Numbers indicate the RCA exons. The forward
and reverse primers for amplification of the 1.4 kb PCR products of
RCA (RCAf and RCAr) and .DELTA.rca (rcaf and rcar) alleles are
indicated. (B) PCR analysis (RCA primers-upper panel; rca
primers-bottom panel) of T1 plants expressing 183H12. Lines number
(up) and genetic background (down) are indicated. (C) Western blot
analysis of total protein (5 .mu.g/lane) from leaves of the lines
described in (B). The blot was probed with polyclonal antibodies
raised against the recombinant RCA1.
[0015] FIGS. 4A-4D: Functional complementation of .DELTA.rca
mutants expressing RCA1 and thermostable variants 183H12, 301C7 and
382D8 under normal growth conditions (22.degree. C.). Numbers
indicate the line designations of independent transformation
events. (A) Immuno blot analysis of total protein from three-week
old leaves. (B) Photographs depicting the similar size of all the
plants described above when grown under normal conditions. (C)
Photosynthetic performance of the plants (8 to 10
plants/independent line) described above monitored by fluorescence
image analysis. (D) Effect of temporary (1 hr) moderate heat stress
treatment on photosynthetic rates of .DELTA.rca transgenic lines
expressing RCA1 and thermostable variants. The net photosynthesis
of four independent plants per line was monitored using an infrared
gas analyzer at 22.degree. C. (white) and 30.degree. C. (gray).
[0016] FIGS. 5A-5F: Effect of moderate heat stress (30.degree. C.
for 4 hr per day) on wild type plants and .DELTA.rca mutants
expressing RCA1 or thermostable variants 183H12, 301C7 and 382D8.
(A) Photograph of the plants showing differential growth rates
mediated by the RCA variant. (B) Leaf area of 8-10 independent
plants per line, analyzed using a fluorescence image analysis
system. Means followed by common letters are not significantly
different at P=0.05 using a protected LSD. (C) Net photosynthesis
of four independent plants from selected lines, monitored by gas
exchange analysis after 2 hr at 30.degree. C. (D) Photograph of
mature plants (8-weeks old) described in (C). (E) Number of
siliques per plant. Eight to ten independent plants per line were
analyzed. (F) Seed weight/1000 seeds of seeds harvested from the
selected lines (5 independent plants per line).
[0017] FIGS. 6A-6D: Effect of 26.degree. C. heat stress on
development and yield of wild type plants and .DELTA.rca mutant
lines expressing RCA1 and thermostable variant 183H12. (A) Number
of siliques per plant. Ten to twelve independent plants per line
were analyzed. (B) Photograph of siliques from selected lines grown
at the indicated temperature showing variation in siliqe size and
seed set. Bar=0.5 cm. (C) Seed weight/1000 seeds for seeds
harvested from the plants described in (A). (D) Germination rates
(at 22.degree. C.) of seeds (250 seeds per line) harvested from
Arabidopsis plants that were grown at 22.degree. C. (white) or
26.degree. C. (gray).
DETAILED DESCRIPTION
[0018] The present invention provides polypeptides derived from
Rubisco Activase. Nucleic acid molecules encoding the polypeptides
of the invention are also provided. Methods for using the
polypeptides and nucleic acids of the invention to increase the
heat-tolerance of plants comprising enhancing the thermostability
of Rubisco Activase are encompassed.
Polypeptides of the Invention
[0019] The present invention relates to Rubisco Activase derived
polypeptides that are more thermostable than naturally occurring
Rubisco Activase. In preferred embodiments, the Rubisco Activase
derived polypeptide is any of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16,
18, 20 and 22. Polypeptides of the invention also encompass those
polypeptides that are encoded by any Rubisco Activase derived
nucleic acid of the invention.
[0020] In addition to the Rubisco Activase derived polypeptides of
the invention, it will be appreciated that the invention also
encompasses variants thereof, including, but not limited to, any
substantially similar sequence, any fusion polypeptide, any
fragment, analog, homolog, mutant or modified polypeptide thereof.
The variants encompassed by the invention are at least partially
functionally active (i.e., they are capable of displaying one or
more known functional activities associated with wild type Rubisco
Activase) under heated conditions. Such functional activities
include, but are not limited to, biological activities, such as
activation of Rubisco; antigenicity, i.e., an ability to bind or
compete with wild type Rubisco Activase (including, but not limited
to, SEQ ID NO: 2) for binding to an anti-Rubisco Activase antibody;
immunogenicity, i.e., an ability to generate antibody which binds
to a wild type Rubisco Activase polypeptide. In preferred
embodiments, the variants have at least one functional activity
that is substantially similar to or better than its parent
polypeptide (i.e., the unaltered Rubisco Activase derived
polypeptide). As used herein, the functional activity of the
variant will be considered "substantially similar" to its parent
polypeptide if it is within one standard deviation of the
parent.
[0021] In one embodiment, polypeptides that have at least one
functional activity of Rubisco Activase (e.g., Rubisco activation)
under heated conditions and are at least 85%, 90%, 95%, 97%, 98% or
99% identical to the polypeptide sequence of any of SEQ ID NOS: 4,
6, 8, 10, 12, 14, 16, 18, 20 and 22 are encompassed by the
invention. In specific embodiments, such polypeptides of the
invention are altered at one or more, two or more, five or more, or
seven or more positions corresponding to residues 42, 130, 131,
168, 257, 274, 293 and 310 of SEQ ID NO: 2 upon optimal alignment
of the polypeptide sequence with SEQ ID NO: 2. With respect to an
amino acid sequence that is optimally aligned with a reference
sequence, an amino acid "corresponds" to the position in the
reference sequence with which the residue is paired in the
alignment.
[0022] As used herein, where a sequence is defined as being "at
least X % identical" to a reference sequence, e.g., "a polypeptide
at least 95% identical to SEQ ID NO: 4," it is to be understood
that "X% identical" refers to absolute percent identity, unless
otherwise indicated. The term "absolute percent identity" refers to
a percentage of sequence identity determined by scoring identical
amino acids or nucleic acids as one and any substitution as zero,
regardless of the similarity of mismatched amino acids or nucleic
acids. In a typical sequence alignment the "absolute percent
identity" of two sequences is presented as a percentage of amino
acid or nucleic acid "identities." In cases where an optimal
alignment of two sequences requires the insertion of a gap in one
or both of the sequences, an amino acid residue in one sequence
that aligns with a gap in the other sequence is counted as a
mismatch for purposes of determining percent identity. Gaps can be
internal or external, i.e., a truncation. Absolute percent identity
can be readily determined using, for example, the Clustal W
program, version 1.8, June 1999, using default parameters
(Thompson, et al., (1994) Nucleic Acids Research 22:4673-4680).
[0023] In another embodiment, fusion polypeptides comprising a
Rubisco Activase derived polypeptide or variant thereof are
encompassed by the invention. In a specific embodiment, a peptide
(such as those disclosed in U.S. patent application Ser. No.
11/150,054) is added onto a polypeptide of the invention, whereby
the peptide directs localization of the attached polypeptide to the
plant plastids or the plant photosynthetic organs.
[0024] In another embodiment, fragments of Rubisco Activase derived
polypeptides are encompassed by the invention. Polypeptides are
encompassed that have at least one functional activity (e.g.,
Rubisco activation) of Rubisco Activase under heated conditions and
are at least 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375 or 380 contiguous amino acids in length of any
of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, and 22. In preferred
embodiments, the polynucleotide that encodes the fragment
polypeptide hybridizes under stringent conditions to the nucleic
acid that encodes any of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18,
20 and 22.
[0025] In a specific embodiment, a fragment of the invention
corresponds to a functional domain of Rubisco Activase including,
but not limited to, the ATP binding domain and the substrate
interaction domain (see, e.g., Li, et al., (2005) J Biol Chem
280:24864-24869; Salvucci, et al., (1994) Biochemistry
33:14879-14886 and van de Loo, et al., (1996) Biochemistry
35:8143-8148).
[0026] In another embodiment, analog polypeptides are encompassed
by the invention. Analog polypeptides may possess residues that
have been modified, i.e., by the covalent attachment of any type of
molecule to the Rubisco Activase derived polypeptides. For example,
but not by way of limitation, an analog polypeptide of the
invention may be modified, e.g., by glycosylation, acetylation,
pegylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. An analog polypeptide of the
invention may be modified by chemical modifications using
techniques known to those of skill in the art, including, but not
limited to specific chemical cleavage, acetylation, formylation,
metabolic synthesis of tunicamycin, etc. Furthermore, an analog of
a polypeptide of the invention may contain one or more
non-classical amino acids.
[0027] In another embodiment of the invention, a Rubisco Activase
derived polypeptide is not SEQ ID NO: 2. In yet another embodiment
of the invention, a Rubisco Activase derived polypeptide is not a
naturally occurring wild type polypeptide.
[0028] Methods of production of the polypeptides of the invention,
e.g., by recombinant means, are also provided.
Nucleic Acid Molecules of the Invention
[0029] The present invention also relates to the nucleic acid
molecules encoding Rubisco Activase derived polypeptides. In
preferred embodiments, the Rubisco Activase derived nucleic acid
molecule is any of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19 and
21. Nucleic acid molecules of the invention also encompass those
nucleic acid molecules that encode any Rubisco Activase derived
polypeptides of the invention.
[0030] In addition to the nucleic acid molecules encoding Rubisco
Activase derived polypeptides, it will be appreciated that nucleic
acid molecules of the invention also encompass those encoding
polypeptides that are variants of Rubisco Activase derived
polypeptides, including, but not limited to any substantially
similar sequence, any fusion polypeptide, any fragment, analog,
homolog, mutant or modified polypeptide thereof. The nucleic acid
molecule variants encompassed by the invention encode polypeptides
that are at least partially functionally active (i.e., they are
capable of displaying one or more known functional activities
associated with wild type Rubisco Activase) under heated
conditions.
[0031] In one embodiment, nucleic acid molecules that are at least
70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any of
the nucleic acid molecules of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15,
17, 19 and 21 are encompassed by the invention. In specific
embodiments, such nucleic acid molecules of the invention encode
polypeptides that are altered at one or more, two or more, five or
more, or seven or more positions corresponding to residues 42, 130,
131, 168, 257, 274, 293 and 310 of SEQ ID NO: 2 upon optimal
alignment of the nucleotide sequence with SEQ ID NO: 2.
[0032] To determine the percent identity of two nucleic acid
molecules, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in the sequence of a first
nucleic acid molecule for optimal alignment with a second or
nucleic acid molecule). The nucleotides at corresponding nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same nucleotide as the corresponding position in
the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=number of identical overlapping
positions/total number of positions .times.100%). In one
embodiment, the two sequences are the same length.
[0033] The determination of percent identity between two sequences
can also be accomplished using a mathematical algorithm. A
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. 87:2264-2268,
modified as in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci.
90:5873-5877). Such an algorithm is incorporated into the NBLAST
and XBLAST programs (Altschul, et al., (1990) J. Mol. Biol. 215:403
and Altschul, et al., (1997) Nucleic Acid Res. 25:3389-3402).
Software for performing BLAST analyses is publicly available, e.g.,
through the National Center for Biotechnology Information. This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul, et al., supra). These initial neighborhood
word hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For polypeptides, a scoring
matrix is used to calculate the cumulative score. Extension of the
word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=.sup.-4 and a comparison of both
strands. For polypeptides, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10 and the BLOSUM62
scoring matrix (see, Henikoff and Henikoff, (1989) PNAS
89:10915).
[0034] The Clustal V method of alignment can also be used to
determine percent identity (Higgins and Sharp, (1989) CABIOS
5:151-153) and found in the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). The
"default parameters" are the parameters pre-set by the manufacturer
of the program and for multiple alignments they correspond to GAP
PENALTY=10 and GAP LENGTH PENALTY=10, while for pairwise alignments
they are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
After alignment of the sequences, using the Clustal V program, it
is possible to obtain a "percent identity" by viewing the "sequence
distances" table on the same program.
[0035] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
[0036] In another embodiment, fragments of Rubisco Activase derived
nucleic acid molecules are encompassed by the invention. Nucleic
acid molecules are encompassed that have at least one functional
activity of Rubisco Activase (e.g., Rubisco activation) under
heated conditions and are at least 100, 250, 500, 750, 950, 1000 or
1100 contiguous nucleotides in length of any of SEQ ID NOS: 3, 5,
7, 9, 11, 13, 15, 17, 19 and 21.
[0037] In a specific embodiment, a fragment of the invention
corresponds to a nucleic acid molecule that encodes a functional
domain of Rubisco Activase including, but not limited to, the ATP
binding domain and the substrate interaction domain (see, e.g., Li,
et al., (2005) J Biol Chem 280:24864-24869; Salvucci, et al.,
(1994) Biochemistry 33:14879-14886 and van de Loo, et al., (1996)
Biochemistry 35:8143-8148).
[0038] In another embodiment, a nucleic acid molecule that
hybridizes under stringent conditions to any one of SEQ ID NOS: 3,
5, 7, 9, 11, 13, 15, 17, 19 and 21 is encompassed by the invention.
The phrase "stringent conditions" refers to hybridization
conditions under which a nucleic acid will hybridize to its target
nucleic acid, typically in a complex mixture of nucleic acid, but
to essentially no other nucleic acids. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer nucleic acids hybridize specifically at
higher temperatures. Extensive guides to the hybridization of
nucleic acids can be found in the art (e.g., Tijssen, Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic
Probes, "Overview of principles of hybridization and the strategy
of nucleic acid assays" (1993)). Generally, highly stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific nucleic acid at a
defined ionic strength pH. Low stringency conditions are generally
selected to be about 15-30.degree. C. below the T.sub.m. The
T.sub.m is the temperature (under defined ionic strength, pH, and
nucleic acid concentration) at which 50% of the probes
complementary to the target hybridize to the target nucleic acid at
equilibrium (as the target nucleic acids are present in excess, at
T.sub.m, 50% of the probes are occupied at equilibrium).
Hybridization conditions are typically those in which the salt
concentration is less than about 1.0 M sodium ion, typically about
0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0
to 8.3 and the temperature is at least about 30.degree. C. for
short probes (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for long probes (e.g., greater than 50 nucleotides).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
and preferably 10 times background hybridization. The phrase
"specifically hybridizes" refers to the binding, duplexing, or
hybridizing of a molecule only to a particular nucleotide sequence
under stringent hybridization conditions when that sequence is
present in a complex mixture (e.g., total cellular or library DNA
or RNA).
[0039] In another embodiment of the invention, a Rubisco Activase
derived nucleic acid molecule is not SEQ ID NO: 1. In yet another
embodiment of the invention, a Rubisco Activase derived nucleic
acid molecule is not a naturally occurring wild type nucleic acid
molecule.
[0040] Vectors comprising nucleic acid molecules of the invention
are also encompassed. Cells or plants comprising the vectors of the
invention are also encompassed.
[0041] The term "nucleic acid molecule" herein refers to a single
or double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. It includes chromosomal DNA,
self-replicating plasmids and DNA or RNA that performs a primarily
structural role.
Rubisco Activase-Derived Sequences
[0042] Rubisco Activase derived polypeptides and nucleic acid
molecules of the invention can be created by introducing one or
more residue substitutions, additions and/or deletions into a wild
type (wt) Rubisco Activase (including, but not limited to,
Arabidopsis Rubisco Activase (SEQ ID NO: 2)). Generally, Rubisco
Activase derived polypeptides are created in order to accentuate a
desirable characteristic or reduce an undesirable characteristic of
a wild type Rubisco Activase polypeptide. In one embodiment,
Rubisco Activase derived polypeptides have improved thermostability
over wild type Rubisco Activase. In another embodiment, Rubisco
Activase derived polypeptides have enzymatic activity under heated
conditions (e.g., 40.degree. C.) that is similar to or higher than
the enzyme activity of wild type Rubisco Activase under normal
conditions (e.g., 25.degree. C.).
[0043] In one embodiment, a wild type Rubisco Activase nucleic acid
molecule (e.g., SEQ ID NO: 1) is used as a template to create
Rubisco Activase derived nucleic acid molecules. In some
embodiments, nucleic acid residues that encode one or more amino
acid residues corresponding to residues 42, 130, 131, 168, 257,
274, 293 and 310 of SEQ ID NO: 2 upon optimal alignment of the
nucleotide sequence with SEQ ID NO: 2 are altered such that the
encoded amino acid is altered.
[0044] Sequence alterations can be introduced by standard
techniques such as directed molecular evolution techniques e.g.,
DNA shuffling methods (see, e.g., Christians, et al., (1999) Nature
Biotechnology 17:259-264; Crameri, et al., (1998) Nature
391:288-291; Crameri, et al., (1997) Nature Biotechnology
15:436-438; Crameri, et al., (1996) Nature Biotechnology
14:315-319; Stemmer, (1994) Nature 370:389-391; Stemmer, et al.,
(1994) Proc. Natl. Acad. Sci. 91:10747-10751; U.S. Pat. Nos.
5,605,793; 6,117,679; 6,132,970; 5,939,250; 5,965,408; 6,171,820;
International Publication Numbers WO 95/22625; WO 97/0078; WO
97/35966; WO 98/27230; WO 00/42651 and WO 01/75767); site directed
mutagenesis (see, e.g., Kunkel, (1985) Proc. Natl. Acad. Sci.
82:488-492; Oliphant, et al., (1986) Gene 44:177-183);
oligonucleotide-directed mutagenesis (see, e.g., Reidhaar-Olson, et
al., (1988) Science 241:53-57); chemical mutagenesis (see, e.g.,
Eckert, et al., (1987) Mutat. Res. 178:1-10); error prone PCR (see,
e.g., Caldwell and Joyce, (1992) PCR Methods Applic. 2:28-33) and
cassette mutagenesis (see, e.g., Arkin, et al., (1992) Proc. Natl.
Acad. Sci., 89:7871-7815); (see generally, e.g., Arnold, (1993)
Curr. Opinion Biotechnol. 4:450-455; Ling, et al., (1997) Anal.
Biochem., 254(2):157-78; Dale, et al., (1996) Methods Mol. Biol.
57:369-74; Smith, (1985) Ann. Rev. Genet. 19:423-462; Botstein, et
al., (1985) Science, 229:1193-1201; Carter, (1986) Biochem. J.
237:1-7; Kramer, et al., (1984) Cell 38:879-887; Wells, et al.,
(1985) Gene 34:315-323; Minshull, et al., (1999) Current Opinion in
Chemical Biology 3:284-290).
[0045] In one embodiment, DNA shuffling is used to create Rubisco
Activase derived nucleic acid molecules. DNA shuffling can be
accomplished in vitro, in vivo, in silico or a combination thereof.
In silico methods of recombination can be affected in which genetic
algorithms are used in a computer to recombine sequence strings
which correspond to homologous (or even non-homologous) nucleic
acids. The resulting recombined sequence strings are optionally
converted into nucleic acids by synthesis of nucleic acids which
correspond to the recombined sequences, e.g., in concert with
oligonucleotide synthesis gene reassembly techniques. This approach
can generate random, partially random or designed alterations. Many
details regarding in silico recombination, including the use of
genetic algorithms, genetic operators and the like in computer
systems, combined with generation of corresponding nucleic acids as
well as combinations of designed nucleic acids (e.g., based on
cross-over site selection) as well as designed, pseudo-random or
random recombination methods are described in the art (see, e.g.,
International Publication Numbers WO 00/42560 and WO 00/42559).
[0046] In another embodiment, targeted mutagenesis is used to
create Rubisco Activase derived nucleic acid molecules by choosing
particular nucleotide sequences or positions of the wild type
Rubisco Activase for alteration. Such targeted mutations can be
introduced at any position in the nucleic acid and can be
conservative or non-conservative.
[0047] A "non-conservative amino acid substitution" is one in which
the amino acid residue is replaced with an amino acid residue
having a dissimilar side chain. Families of amino acid residues
having similar side chains have been defined in the art. These
families include amino acids with basic side chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid, asparagine, glutamine), uncharged polar side chains
(e.g., glycine, serine, threonine, tyrosine, cysteine), nonpolar
side chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), .beta.-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0048] Alternatively or in addition to non-conservative amino acid
residue substitutions, such targeted mutations can be conservative.
A "conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Following mutagenesis, the encoded protein can
be expressed recombinantly and the activity of the protein can be
determined.
[0049] In some embodiments, substitutions can be made such that the
amino acid at position 42 has an uncharged polar side chain or a
.beta.-branched side chain, at position 130 has a basic side chain,
at position 131 has a nonpolar side chain or a .beta.-branched side
chain, at position 168 has a basic side chain, at position 257 has
a nonpolar side chain or a .beta.-branched side chain, at position
274 has a basic side chain, at position 293 has a basic side chain
and/or at position 310 has an acidic side chain. The amino acid
positions can be determined by optimally aligning the amino acid
sequence of the encoded Rubisco Activase derived polypeptide with
SEQ ID NO: 2.
[0050] In another embodiment, random mutagenesis is used to create
Rubisco Activase derived nucleic acid molecules. Mutations can be
introduced randomly along all or part of the coding sequence (e.g.,
by saturation mutagenesis). In certain embodiments, nucleotide
sequences encoding other related polypeptides that have similar
domains, structural motifs, active sites, or that align with a
portion of wild type Rubisco Activase with mismatches or imperfect
matches, can be used in the mutagenesis process to generate
diversity of sequences.
[0051] It should be understood that for each mutagenesis step in
some of the techniques mentioned above, a number of iterative
cycles of any or all of the steps may be performed to optimize the
diversity of sequences. The above-described methods can be used in
combination in any desired order. In many instances, the methods
result in a pool of altered nucleic acid sequences or a pool of
recombinant host cells comprising altered nucleic acid sequences.
The altered nucleic acid sequences or host cells expressing an
altered nucleic acid sequence with the desired characteristics can
be identified by screening with one or more assays known in the
art. The assays may be carried out under conditions that select for
polypeptides possessing the desired physical or chemical
characteristics. The alterations in the nucleic acid sequence can
be determined by sequencing the nucleic acid molecule encoding the
altered polypeptide in the clones.
[0052] Additionally, Rubisco Activase derived nucleic acid
molecules can be codon optimized, either wholly or in part. Because
any one amino acid (except for methionine) is encoded by a number
of codons (Table 1), the sequence of the nucleic acid molecule may
be changed without changing the encoded amino acid. Codon
optimization is when one or more codons are altered at the nucleic
acid level to coincide with or better approximate the codon usage
of a particular host. The frequency of preferred codon usage
exhibited by a host cell can be calculated by averaging frequency
of preferred codon usage in a large number of genes expressed by
the host cell. This analysis may be limited to genes that are
highly expressed by the host cell. U.S. Pat. No. 5,824,864, for
example, provides the frequency of codon usage by highly expressed
genes exhibited by dicotyledonous plants and monocotyledonous
plants. Those having ordinary skill in the art will recognize that
tables and other references providing preference information for a
wide range of organisms are available in the art.
Methods of Assaying Rubisco Activase Activity
[0053] The present invention is directed to Rubisco Activase
derived polypeptides with improved thermostability as compared to
wild type Rubisco Activase. As used herein, the term "improved
thermostability" refers to the increased ability of Rubisco
Activase to activate Rubisco under heated conditions as compared to
wild type Rubisco Activase. In one embodiment, Rubisco Activase
derived polypeptides have enzymatic activity under heated
conditions (e.g., 35.degree. C. or higher) that is greater than the
enzymatic activity of wild type Rubisco Activase under heated
conditions. In another embodiment, Rubisco Activase derived
polypeptides have enzymatic activity under heated conditions (e.g.,
26.degree. C. or higher, more preferably 40.degree. C. for in vitro
assays) that is substantially similar to or higher than the enzyme
activity of wild type Rubisco Activase under normal conditions
(e.g., 20-25.degree. C., more preferably 25.degree. C. for in vitro
assays and 22.degree. C. for in vivo assays). As used herein, the
term "substantially similar" refers to enzymatic activity of a
Rubisco Activase derived polypeptide that is within one standard
deviation of that of wild type Rubisco.
[0054] Any method known in the art can be used to assay the
activity of Rubisco Activase derived polypeptides (including, but
not limited to, Rubisco activation and ATP hydrolysis) under heated
conditions.
[0055] In some embodiments, Rubisco Activase derived polypeptide
activity is assayed in vitro. In one embodiment, Rubisco Activase
derived polypeptides can be assayed for their ability to activate
Rubisco when incubated in a solution comprising deactivated
Rubisco, RuBP, ATP and a source of labeled carbon (e.g.,
[C.sup.14]NaHCO.sub.3). Incorporation of labeled carbon into
3-phosphoglyceric acid (GPA) can be monitored as an indication of
Rubisco activation. In another embodiment, Rubisco Activase derived
polypeptides can be assayed for their ability to hydrolyze ATP when
incubated in a solution comprising ATP. The assays can be conducted
under heated conditions or under normal conditions after the
Rubisco Activase derived polypeptides are heat treated prior to
performance of the assay. Purified components can be used as well
as cells comprising the components for Rubisco activation.
[0056] In other embodiments, Rubisco Activase derived polypeptide
activity is assayed in vivo. Plants which do not express wild type
Rubisco Activase (e.g., deletion mutants) are made to express one
or more Rubisco Activase derived polypeptides and can be analyzed
for photosynthesis rates, biomass, growth rates and seed yield
under heated growing conditions. The plants may be grown entirely
under heated conditions or for shorter periods of time under heated
conditions. In one embodiment, photosynthesis rates are measured by
analyzing the plants for CO.sub.2 fixation. In another embodiment,
growth rates are measured by analyzing leaf area of the plants. In
another embodiment, seed yield is analyzed by determining seed
weight from mature dried plants. In another embodiment, seed yield
is analyzed by determining the seed germination rate.
Methods of Enhancing Heat Tolerance in Plants
[0057] Rubisco Activase activates Rubisco in plants. Activated
Rubisco is involved in photosynthesis and is the rate limiting step
of the photosynthetic process. With decreased Rubisco Activase
activity, Rubisco remains inactive and photosynthesis slows or
stops. Increased temperatures destabilize and/or denature Rubisco
Activase thereby rendering the enzyme less able or unable to
convert inactive Rubisco to the active form. The present invention
is directed to Rubisco Activase derived polypeptides with improved
thermostability as compared to wild type Rubisco Activase. As such,
the photosynthetic process can be made more heat tolerant with the
involvement of Rubisco Activase derived polypeptides with improved
thermostability.
[0058] Any method known in the art can be used to cause plants to
express one or more of the Rubisco Activase derived polypeptides of
the invention. In one embodiment, transgenic plants can be made to
express one or more polypeptides of the invention. The transgenic
plant may express the one or more polypeptides of the invention in
all tissues (e.g., global expression). Alternatively, the one or
more polypeptides of the invention may be expressed in only a
subset of tissues (e.g., tissue specific expression), preferably
those tissues or organelles involved in photosynthesis (e.g., the
plastids). Polypeptides of the invention can be expressed
constitutively in the plant or be under the control of an inducible
promoter. In some embodiments, the expression and/or activity of
the endogenous Rubisco Activase of the plant is reduced or
eliminated.
Recombinant Expression
[0059] Nucleic acid molecules and polypeptides of the invention can
be expressed recombinantly using standard recombinant DNA and
molecular cloning techniques that are well known in the art (e.g.,
Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory
Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor,
(1989)). Additionally, recombinant DNA techniques may be used to
create nucleic acid constructs suitable for use in making
transgenic plants.
[0060] Accordingly, an aspect of the invention pertains to vectors,
preferably expression vectors, comprising a nucleic acid molecule
of the invention or a variant thereof. As used herein, the term
"vector" refers to a polynucleotide capable of transporting another
nucleic acid to which it has been linked. One type of vector is a
"plasmid", which refers to a circular double stranded DNA loop into
which additional DNA segments can be introduced. Another type of
vector is a viral vector, wherein additional DNA segments can be
introduced into the viral genome.
[0061] Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal vectors).
Other vectors (e.g., non-episomal vectors) are integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids (vectors). However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses).
[0062] The recombinant expression vectors of the invention comprise
a nucleic acid molecule of the invention in a form suitable for
expression of the nucleic acid molecule in a host cell. This means
that the recombinant expression vectors include one or more
regulatory sequences, selected on the basis of the host cells to be
used for expression, which is operably associated with the
polynucleotide to be expressed. Within a recombinant expression
vector, "operably associated" is intended to mean that the
nucleotide sequence of interest is linked to the regulatory
sequence(s) in a manner which allows for expression of the
nucleotide sequence (e.g., in an in vitro transcription/translation
system or in a host cell when the vector is introduced into the
host cell). The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described
in the art (e.g., Goeddel, Gene Expression Technology: Methods in
Enzymology, (1990) Academic Press, San Diego, Calif.). Regulatory
sequences include those which direct constitutive expression of a
nucleotide sequence in many types of host cells and those which
direct expression of the nucleotide sequence only in certain host
cells (e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, the area of the organism in which expression is desired,
etc. The expression vectors of the invention can be introduced into
host cells to thereby produce proteins or peptides, including
fusion proteins or peptides, encoded by nucleic acids molecules as
described herein.
[0063] In some embodiments, isolated nucleic acids which serve as
promoter or enhancer elements can be introduced in the appropriate
position (generally upstream) of a non-heterologous form of a
polynucleotide of the present invention so as to up or down
regulate expression of a polynucleotide of the present invention.
For example, endogenous promoters can be altered in vivo by
mutation, deletion and/or substitution (see, U.S. Pat. No.
5,565,350; International Patent Application Number PCT/US93/03868)
or isolated promoters can be introduced into a plant cell in the
proper orientation and distance from a cognate gene of a
polynucleotide of the present invention so as to control the
expression of the gene. Gene expression can be modulated under
conditions suitable for plant growth so as to alter the total
concentration and/or alter the composition of the polypeptides of
the present invention in plant cell. Thus, the present invention
provides compositions, and methods for making heterologous
promoters and/or enhancers operably linked to a native, endogenous
(i.e., non-heterologous) form of a polynucleotide of the present
invention.
[0064] If polypeptide expression is desired, it is generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region. The polyadenylation region can be
derived from the natural gene, from a variety of other plant genes,
or from T-DNA. The 3' end sequence to be added can be derived from,
for example, the nopaline synthase or octopine synthase genes, or
alternatively from another plant gene or less preferably from any
other eukaryotic gene.
[0065] The recombinant expression vectors of the invention can be
designed for expression of a polypeptide of the invention in
prokaryotic (e.g., Enterobacteriaceae, such as Escherichia;
Bacillaceae; Rhizoboceae, such as Rhizobium and Rhizobacter;
Spirillaceae, such as photobacterium; Zymomonas; Serratia;
Aeromonas; Vibrio; Desulfovibrio; Spirillum; Lactobacillaceae;
Pseudomonadaceae, such as Pseudomonas and Acetobacter,
Azotobacteraceae and Nitrobacteraceae) or eukaryotic cells (e.g.,
insect cells using baculovirus expression vectors, yeast cells,
plant cells or mammalian cells) (see, Goeddel, supra. for a
discussion on suitable host cells). Alternatively, the recombinant
expression vector can be transcribed and translated in vitro, for
example using T7 promoter regulatory sequences and T7
polymerase.
[0066] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors comprising constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve at least three
purposes: 1) to increase expression of the recombinant protein; 2)
to increase the solubility of the recombinant protein and/or 3) to
aid in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, (1988) Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein or protein A, respectively, to the target
recombinant protein.
[0067] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz, et al., (1987) Gene 54:113-123), pYES2 (Invitrogen
Corp., San Diego, Calif.), and pPicZ (Invitrogen Corp., San Diego,
Calif.).
[0068] Alternatively, the expression vector is a baculovirus
expression vector. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith, et al., (1983) Mol. Cell. Biol. 3:2156-2165) and
the pVL series (Lucklow and Summers, (1989) Virology
170:31-39).
[0069] In yet another embodiment, a nucleic acid molecule of the
invention is expressed in plant cells using a plant expression
vector including, but not limited to, tobacco mosaic virus and
potato virus expression vectors.
[0070] Other suitable expression systems for both prokaryotic and
eukaryotic cells are known in the art (see, e.g., chapters 16 and
17 of Sambrook, et al., (1990) Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0071] A number of promoters can be used in the practice of the
invention. The promoters can be selected based on the desired
outcome. The nucleic acids can be combined with constitutive,
tissue-specific, inducible or other promoters for expression in the
host organism.
[0072] A "tissue-specific promoter" may direct expression of
nucleic acids of the present invention in a specific tissue, organ
or cell type. Tissue-specific promoters can be inducible.
Similarly, tissue-specific promoters may only promote transcription
within a certain time frame or developmental stage within that
tissue. Other tissue specific promoters may be active throughout
the life cycle of a particular tissue. One of ordinary skill in the
art will recognize that a tissue-specific promoter may drive
expression of operably linked sequences in tissues other than the
target tissue. Thus, as used herein, a tissue-specific promoter is
one that drives expression preferentially in the target tissue or
cell type, but may also lead to some expression in other tissues as
well. A number of tissue-specific promoters can be used in the
present invention. With the appropriate promoter, any organ can be
targeted, such as shoot vegetative organs/structures (e.g., leaves,
stems and tubers), roots, flowers and floral organs/structures
(e.g., bracts, sepals, petals, stamens, carpels, anthers and
ovules), seed (including embryo, endosperm and seed coat) and
fruit. For instance, promoters that direct expression of nucleic
acid molecules in leaves and/or photosynthetic organ-specific
promoters (such as the RBCS promoter disclosed in Khoudi, et al.,
(1997) Gene 197:343) are useful for enhancing photosynthesis.
Additionally, tissue specific expression can be obtained by adding
a peptide onto a polypeptide of the invention that directs
localization of the attached polypeptide to the photosynthetic
organs (such as those disclosed in U.S. patent application Ser. No.
11/150,054).
[0073] A "constitutive promoter" is defined as a promoter which
will direct expression of a gene in all tissues and are active
under most environmental conditions and states of development or
cell differentiation. Examples of constitutive promoters include
the cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumafaciens, and other transcription initiation regions from
various plant genes known to those of ordinary skill in the art.
Such genes include for example, ACT11 from Arabidopsis (Huang, et
al., (1996) Plant Mol. Biol. 33:125-139), Cat3 from Arabidopsis
(GenBank Accession Number U43147, Zhong, et al., (1996) Mol. Gen.
Genet. 251:196-203), the gene encoding stearoyl-acyl carrier
protein desaturase from Brassica napus (Genbank Accession Number
X74782, Solocombe, et al., (1994) Plant Physiol. 104:1167-1176),
GPc1 from maize (GenBank Accession Number X15596, Martinez, et al.,
(1989) J. Mol. Biol. 208:551-565), and Gpc2 from maize (GenBank
Accession Number U45855, Manjunath, et al., (1997) Plant Mol. Biol.
33:97-112). Any strong, constitutive promoter, such as the CaMV 35S
promoter, can be used for the expression of polynucleotides of the
present invention throughout the plant.
[0074] The term "inducible promoter" refers to a promoter that is
under precise environmental or developmental control. Examples of
environmental conditions that may effect transcription by inducible
promoters include anaerobic conditions, elevated temperature, the
presence of light or spraying with chemicals/hormones.
[0075] Suitable constitutive promoters for use in a plant host cell
include, for example, the core promoter of the Rsyn7 promoter and
other related constitutive promoters (International Publication
Number WO 99/43838 and U.S. Pat. No. 6,072,050); the core CaMV 35S
promoter (Odell, et al., (1985) Nature 313:810-812); rice actin
(McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin
(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and
Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU
(Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten,
et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.
5,659,026) and the like (e.g., U.S. Pat. Nos. 5,608,149; 5,608,144;
5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142
and 6,177,611).
[0076] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0077] Accordingly, the present invention provides a host cell
having an expression vector comprising a nucleic acid molecule of
the invention, or a variant thereof. A host cell can be any
prokaryotic (e.g., E. coli, Bacillus thuringiensis) or eukaryotic
cell (e.g., insect cells, yeast or plant cells). The invention also
provides a method for expressing a nucleic acid molecule of the
invention thus making the encoded polypeptide comprising the steps
of i) culturing a cell comprising a nucleic acid molecule of the
invention under conditions that allow production of the encoded
polypeptide; and ii) isolating the expressed polypeptide.
[0078] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid molecules into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, electroporation,
Agrobacterium tumefaciens and vacuum infiltration. Suitable methods
for transforming or transfecting host cells can be found in the art
(e.g., Sambrook, et al., supra.).
Production of Transgenic Plants
[0079] Any method known in the art can be used for transforming a
plant or plant cell with a nucleic acid molecule of the present
invention. Nucleic acid molecules can be incorporated into plant
DNA (e.g., genomic DNA or chloroplast DNA) or be maintained without
insertion into the plant DNA (e.g., through the use of artificial
chromosomes). Suitable methods of introducing nucleic acid
molecules into plant cells include microinjection (Crossway, et
al., (1986) Biotechniques 4:320-334); electroporation (Riggs, et
al., (1986) Proc. Natl. Acad. Sci. 83:5602-5606; D'Halluin, et al.,
(1992) Plant Cell 4:1495-1505); Agrobacterium-mediated
transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840, Osjoda, et
al., (1996) Nature Biotechnology 14:745-750; Horsch, et al., (1984)
Science 233:496-498; Fraley, et al., (1983) Proc. Natl. Acad. Sci.
80:4803; and Gene Transfer to Plants, Potrykus, ed.,
Springer-Verlag, Berlin 1995); direct gene transfer (Paszkowski, et
al., (1984) EMBO J. 3:2717-2722); ballistic particle acceleration
(U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes,
et al., (1995) "Direct DNA Transfer into Intact Plant Cells via
Microprojectile Bombardment, in Plant Cell, Tissue, and Organ
Culture: Fundamental Methods, ed. Gamborg and Phillips,
Springer-Verlag, Berlin; and McCabe, et al., (1988) Biotechnology
6:923-926); virus-mediated transformation (U.S. Pat. Nos.
5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931); pollen
transformation (De Wet, et al., (1985) in The Experimental
Manipulation of Ovule Tissues, ed. Chapman, et al., Longman, N.Y.,
pp. 197-209); Lec 1 transformation (U.S. patent application Ser.
No. 09/435,054; International Patent Publication Number WO
00/28058); whisker-mediated transformation (Kaeppler, et al.,
(1990) Plant Cell Reports 9:415-418; Kaeppler, et al., (1992)
Theor. Appl. Genet. 84:560-566) and chloroplast transformation
technology (Bogorad, (2000) Trends in Biotechnology 18:257-263;
Ramesh, et al., (2004) Methods Mol. Biol. 274:301-7; Hou, et al.,
(2003) Transgenic Res. 12:111-4; Kindle, et al., (1991) Proc. Natl.
Acad. Sci. 88:1721-5; Bateman and Purton, (2000) Mol Gen Genet.
263:404-10; Sidorov, et al., (1999) Plant J. 19:209-216).
[0080] The choice of transformation protocols used for generating
transgenic plants and plant cells can vary depending on the type of
plant or plant cell, i.e., monocot or dicot, targeted for
transformation. Examples of transformation protocols particularly
suited for a particular plant type include those for: potato (Tu,
et al., (1998) Plant Molecular Biology 37:829-838; Chong, et al.,
(2000) Transgenic Research 9:71-78); soybean (Christou, et al.,
(1988) Plant Physiol. 87:671-674; McCabe, et al., (1988)
BioTechnology 6:923-926; Finer and McMullen, (1991) In Vitro Cell
Dev. Biol. 27P:175-182; Singh, et al., (1998) Theor. Appl. Genet.
96:319-324); maize (Klein, et al., (1988) Proc. Natl. Acad. Sci.
85:4305-4309; Klein, et al., (1988) Biotechnology 6:559-563; Klein,
et al., (1988) Plant Physiol. 91:440-444; Fromm, et al., (1990)
Biotechnology 8:833-839; Tomes, et al., (1995) "Direct DNA Transfer
into Intact Plant Cells via Microprojectile Bombardment," in Plant
Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg
(Springer-Verlag, Berlin)); cereals (Hooykaas-Van Slogteren, et
al., (1984) Nature 311:763-764; U.S. Pat. No. 5,736,369).
[0081] In some embodiments, more than one construct is used for
transformation in the generation of transgenic plants and plant
cells. Multiple constructs may be included in cis or trans
positions. In preferred embodiments, each construct has a promoter
and other regulatory sequences.
[0082] The transgenic plants can express the transgene in any way
known in the art including, but not limited to, constitutive
expression, developmentally regulated expression, and tissue
specific expression. In a specific embodiment, promoters that
direct expression of nucleic acid molecules in leaves and/or
photosynthetic organs (such as the RBCS promoter disclosed in
Khoudi, et al., Gene 197:343) are used to express the nucleic acid
molecules and/or polypeptides of the invention.
[0083] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant which possesses the transformed genotype and thus the
desired phenotype. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker that
has been introduced together with the desired nucleotide sequences.
Plant regeneration from cultured protoplasts is described in the
art (e.g., Evans, et al., Protoplasts Isolation and Culture,
Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing
Company, New York, 1983; and Binding, Regeneration of Plants, Plant
Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985). Regeneration
can also be obtained from plant callus, explants, organs or parts
thereof. Such regeneration techniques are also described in the art
(e.g., Klee, et al., (1987) Ann. Rev. of Plant Phys
38:467-486).
[0084] The term "plant" includes whole plants, shoot vegetative
organs/structures (e.g. leaves, stems and tubers), roots, flowers
and floral organs/structures (e.g. bracts, sepals, petals, stamens,
carpels, anthers and ovules), seed (including embryo, endosperm and
seed coat) and fruit (the mature ovary), plant tissue (e.g.
vascular tissue, ground tissue and the like) and cells (e.g. guard
cells, egg cells, trichomes and the like) and progeny of same. The
class of plants that can be used in methods of the present
invention includes the class of higher and lower plants amenable to
transformation techniques, including angiosperms (monocotyledonous
and dicotyledonous plants), gymnosperms, ferns and multicellular
algae. Plants of a variety of ploidy levels, including aneuploid,
polyploid, diploid, haploid and hemizygous plants are also
included.
[0085] The nucleic acid molecules of the invention can be used to
confer desired traits on essentially any plant. Thus, the invention
has use over a broad range of plants, including species from the
genera Agrotis, Allium, Ananas, Anacardium, Apium, Arachis,
Asparagus, Athamantha, Atropa, Avena, Bambusa, Beta, Brassica,
Bromus, Browaalia, Camellia, Cannabis, Carica, Ceratonia. Cicer,
Chenopodium, Chicorium, Citrus, Citrullus, Capsicum, Carthamus,
Cocos, Coffea, Coix, Cucumis, Cucurbita, Cynodon, Dactylis, Datura,
Daucus, Dianthus, Digitalis, Dioscorea, Elaeis, Eliusine,
Euphorbia, Festuca, Ficus, Fragaria, Geranium, Glycine, Graminae,
Gossypium, Helianthus, Heterocallis, Hevea, Hibiscus, Hordeum,
Hyoscyamus, Ipomoea, Lactuca, Lathyrus, Lens, Lilium, Linum,
Lolium, Lotus, Lupinus, Lycopersicon, Macadamia, Macrophylla,
Malus, Mangifera, Manihot, Majorana, Medicago, Musa, Narcissus,
Nemesia, Nicotiana, Onobrychis, Olea, Olyreae, Oryza, Panicum,
Panicum, Panieum, Pannisetum, Pennisetum, Petunia, Pelargonium,
Persea, Pharoideae, Phaseolus, Phleum, Picea, Poa, Pinus,
Pistachia, Pisum, Populus, Pseudotsuga, Pyrus, Prunus, Pseutotsuga,
Psidium, Quercus, Ranunculus, Raphanus, Ribes, Ricinus,
Rhododendron, Rosa, Saccharum, Salpiglossis, Secale, Senecio,
Setaria, Sequoia, Sinapis, Solanum, Sorghum, Stenotaphrum,
Theobromus, Trigonella, Trifolium, Trigonella, Triticum, Tsuga,
Tulipa, Vicia, Vitis, Vigna and Zea.
[0086] In specific embodiments, transgenic plants are maize,
tomato, potato, rice, soybean, cotton, sunflower, alfalfa, lettuce,
canola, sorghum or tobacco plants.
[0087] Transgenic plants may be grown and pollinated with either
the same transformed strain or different strains. Two or more
generations of the plants may be grown to ensure that expression of
the desired nucleic acid molecule, polypeptide and/or phenotypic
characteristic is stably maintained and inherited. One of ordinary
skill in the art will recognize that after the nucleic acid
molecule of the present invention is stably incorporated in
transgenic plants and confirmed to be operable, it can be
introduced into other plants by sexual crossing. Any of a number of
standard breeding techniques can be used, depending upon the
species to be crossed.
Determination of Expression in Transgenic Plants
[0088] Any method known in the art can be used for determining the
level of expression in a plant of a nucleic acid molecule of the
invention or polypeptide encoded therefrom. For example, the
expression level in a plant of a polypeptide encoded by a nucleic
acid molecule of the invention can be determined using molecular
techniques including, but not limited to, immunoassay,
immunoprecipitation, gel electrophoresis and quantitative gel
electrophoresis.
[0089] Additionally, the expression level in a plant of a
polypeptide encoded by a nucleic acid molecule of the invention can
be determined by the degree to which the plant phenotype
(including, but not limited to, photosynthesis rates, growth rates,
and seed yield) is altered under heated conditions compared to
plants expressing wild type Rubisco Activase.
[0090] Furthermore, extracts or polypeptides isolated from
transgenic plants, tissues thereof, or cells thereof can be used in
in vitro assays.
[0091] The contents of all published articles, books, reference
manuals and abstracts cited herein, are hereby incorporated by
reference in their entirety to more fully describe the state of the
art to which the invention pertains.
[0092] As various changes can be made in the above-described
subject matter without departing from the scope and spirit of the
present invention, it is intended that all subject matter contained
in the above description and/or defined in the appended claims, be
interpreted as descriptive and illustrative of the present
invention. Modifications and variations of the present invention
are possible in light of the above teachings.
EXAMPLES
Example 1
Isolating Rubisco Activase Derived Polypeptides
[0093] Rubisco Activase libraries were generated from single gene
shuffling (see, e.g., Crameri, et al., (1998) Nature.
391(6664):288-91; Chang, et al., (1999) Nat. Biotechnol.
17(8):793-7; Ness, et al., (1999) Nat. Biotechnol. 17(9):893-6;
Christians, et al., (1999) Nat. Biotechnol. 17(3):259-64 and U.S.
Pat. Nos. 6,605,430; 6,117,679 and 5,605,793) and synthetic
shuffling (see, e.g., U.S. Pat. No. 6,436,675 and International
Publication Numbers WO 00/42561; WO 01/23401; WO 00/42560; and WO
00/42559) using wild type Rubisco Activase of SEQ ID NO: 1 as a
template.
[0094] Briefly, Arabidopsis RNA was isolated from green leaves
using Trizol.RTM. reagent according to the manufacturer's protocol
(Invitrogen). RCA cDNA (GenBank accession number NM 179990) was PCR
cloned into TOPO.RTM. vector (Invitrogen) using TITANIUM.TM.
one-step RT-PCR Kit (BD Biosciences-Clontech). For single gene
shuffling in the first round, the mature RCA short form (coding
region V59 to K438) was PCR amplified (Qiagen Taq DNA polymerase or
Stratagene Mutazyme DNA polymerase), fragmented and reassembled in
a primerless PCR reaction and the shuffled genes were then rescued
with flanking primers that contain an NcoI site (5') and a BamHI
site, 6.times.-His coding region and a stop codon (3'). The library
of variants was cloned into an E. coli expression vector (pET16b,
Novagen) digested with NcoI and BamHI. To increase pool of genetic
in the first round, synthetic shuffling was carried out using
diversity from wheat, rice, cotton, spinach and cucumber (see,
Ness, et al., (2002) Nat. Biotechnol. 20:1251-1255). A second round
of gene shuffling using first round variants as parents was
performed as previously described by Crameri, et al., (1998) Nature
15:288-291.
[0095] Rubisco Activase derived polypeptides with improved
thermostability were isolated by the following screening
methodology.
[0096] First tier: Rubisco Activation Assay. Cultures of E. coli
expressing a Rubisco Activase derived polypeptide were pelleted at
4.degree. C., 3,500 rpm for 15 minutes and stored in a 96-well
V-bottom PCR-plate at -80.degree. C. E. coli cell lysate was
prepared by thawing the cultures at room temperature for 5 minutes,
adding 75 .mu.l Sonication buffer (100 mM Tricine KOH pH 8.0; 20 mM
ascorbate; 3 mM Mg-ATP; 10 mM MgCl.sub.2; 10% v/v glycerol; 10 mM
.beta.me; x3.33 protease inhibitor; 1 .mu.l/ml benzonase; 1 mg/ml
lysosyme) to each well and shaking the plate for 60 minutes at
4.degree. C. until the pellet was lysed. The plates were sonicated
with MISONIX microplate sonicator for 1 minute and then cooled for
1 minute. This process was repeated four times. The cultures were
centrifuged at 4,000 rpm for 20 minutes at 4.degree. C. The
supernatant that contained soluble protein was used in the Rubisco
activation assays.
[0097] Twenty-two .mu.l of the E. coli supernatant was transferred
to a 96-well U-bottom scintillation-plate and incubated at room
temperature for 15 minutes (and used as the lysate for "normal
conditions" in the assays). Heat treatment of the lysate was
performed by transferring 35 .mu.l of the E. coli supernatant to a
96-well V-bottom PCR-plate and incubating at 40.degree. C. for 15
minutes. The heat treated supernatant was then transferred to a
96-well U-bottom scintillation-plate (and used as the lysate for
"heat treated conditions" in the assays). Both plates were
incubated for 5 minutes at 4.degree. C. before assay
performance.
[0098] Rubisco activation was assayed by incubating the cell lysate
containing Rubisco Activase or a Rubisco Activase derived
polypeptide with purified deactivated Arabidopsis Rubisco (15
.mu.g) in reaction buffer (100 mM Tricine KOH pH 8.0; 10 mM
MgCl.sub.2; 10 mM [.sup.14C]NaHCO.sub.3; Mg-ATP; 4 mM RuBP; 1 mM
PEP; 40 .mu.g/ml pyruvate kinase) at room temperature for 15
minutes (see, Shen, et al., (1991) J. Biol. Chem. 266:8963-8968).
The activation of Rubisco by cell lysate expressing Rubisco
Activase derived polypeptides was terminated by addition of 1 N
HCl, and the incorporation of .sup.14CO.sub.2 determined by liquid
scintillation spectroscopy.
[0099] The Rubisco used in the above-described assay was purified
from Arabidopsis leaves. The leaves were homogenized and frozen in
liquid nitrogen before resuspension in extraction buffer (100 mM
Hepes-KOH pH 8.0; 1 mM EDTA pH 8.0; 3 mM DDT; 0.5 mM PMSF; 10 mM
MgCl.sub.2; 10 mM NaHCO.sub.3). The suspension was centrifuged at
12,000 rpm for 20 min at 4.degree. C. and the supernatant was
collected. The supernatant was kept on ice under continuous
stirring while ammonium sulfate was added to a final concentration
of 35% of saturation and centrifuged at 12,000 rpm for 20 min at
4.degree. C. The supernatant was stirred for an additional 30 min.
with ammonium sulfate to a final concentration of 55% of saturation
before centrifugation at 12,000 rpm for 20 min at 4.degree. C. The
pellet was dissolved in extraction buffer and further precipitated
with 18% polyethylene glycol and centrifugation. The pellet was
resuspended in extraction buffer (about 1 ml/original 40 ml of
supernatant) and centrifuged at 13,000 rpm for 30 min at 4.degree.
C. The purified Rubisco was in the supernatant and glycerol was
added to a final concentration of 10%.
[0100] The purified Rubisco was deactivated by the following
protocol (see, Wang, et al., (1992) Plant Physiol. 100:1858-1862).
Ten mM of DTT was added to the purified Rubisco and incubated at
45.degree. C. for 10 min. One ml of the mixture was added to a 20
ml Sephadex G-50 column equilibrated with equilibration buffer (50
mM Tricine-KOH pH 8.0 and 0.5 mM EDTA pH 8.0). Deactivated Rubisco
was eluted from the column by the addition of 1 ml fractions of
equilibration buffer. The eluted deactivated Rubisco was collected
and incubated at room temperature to 1 hour before incubation on
ice for one hour in the presence of 4 mM RuBp.
[0101] Second tier: HTP temperature profile of Rubisco activation
by cell lysate of active clones. Clones of interest identified in
the first tier screening were further characterized in the second
tier of screening. Cell lysate from each active clone was retested
as described above except that the temperature treatment was
carried out at four different temperatures (16.degree. C.,
25.degree. C., 40.degree. C. and 45.degree. C.) prior to assay.
Clones that possessed relatively improved thermostability profile
compare to wide type Rubisco Activase (SEQ ID NO: 2) were selected
for 3.sup.rd tier screening.
[0102] Third tier: Temperature profile of Rubisco activation by
purified Rubisco Activase variants. In order to determine the
specific activity of the derived polypeptides identified by the
first two tiers of screening, affinity purified polypeptides were
pre-incubated at different temperatures and then analyzed for their
ability to activate Rubisco at 25.degree. C. Since Rubisco Activase
catalyses deactivated Rubisco in a time dependent manner, each
reaction was monitored for 15 minutes in 3-minute intervals. The
ratio of Rubisco Activase:Rubisco was set to 1:40 similarly to the
ratio in plant leaves. The thermostability of wild type Rubisco
Activase (SEQ ID NO: 2) and Rubisco Activase derived polypeptides
is shown in Table 2. The percent thermostability represents the
amount of Rubisco that has been activated by Rubisco Activase at
40.degree. C. as a percent of the amount of Rubisco that has been
activated by Rubisco Activase at 25.degree. C.
Example 2
In vitro characterization of Rubisco Activase Derived
Polypeptides
[0103] The Rubisco Activase derived polypeptides isolated in
Example 6.1 were tested in vitro in three different assays in order
to determine the specific activity at 25.degree. C. and 40.degree.
C. and their thermostability (t.s.). In all cases, the results
obtained with wild type Rubisco Activase at 25.degree. C. were set
to 100%.
[0104] Activation of deactivated Rubisco. Purified Rubisco Activase
derived polypeptides were assayed as described in the first tier
assay of Example 1 except that the derived polypeptides were
incubated at 40.degree. C. or 45.degree. C. for 15, 30, 45 or 60
minutes prior to performance of the Rubisco activation assay.
Results are shown in columns 2-4 of Table 3. Column 2 of Table 3
represents the amount of activated Rubisco that is obtained after
incubation of deactivated Rubisco with Rubisco Activase at
25.degree. C. There were no heated conditions used. Column 3 of
Table 3 represents the amount of Rubisco that has been activated by
Rubisco Activase after a 15 min 40.degree. C. heat treatment as a
percent of the amount of Rubisco that has been activated by Rubisco
Activase at 25.degree. C. Column 4 of Table 3 represents the amount
of Rubisco that has been activated by Rubisco Activase with a 45
min 40.degree. C. heat treatment as a percent of the amount of
Rubisco that has been activated by Rubisco Activase with no heat
treatment (at 25.degree. C.).
[0105] Rubisco activation by Rubisco Activase derived polypeptides
301C7 and 382D8 exhibit high thermostability at 40.degree. C. and
45.degree. C. treatments (FIG. 1A). The activity of 382D8 after
45.degree. C. treatment was 80% higher than RCA1 at the same
temperature treatment and only 10% less than the activity of RCA1
incubated at 25.degree. C.
[0106] Rubisco activation under catalytic conditions. Purified
Rubisco Activase derived polypeptides were assayed as described in
the first tier assay of Example 1 except that the assay was
performed under heated conditions (i.e., 40.degree. C.)
(Crafts-Brandner and Salvucci, (2000) PNAS 97:13430-13435). Results
are shown in columns 5-6 of Table 3. Column 5 of Table 3 represents
the amount of activated Rubisco that is obtained after incubation
of deactivated Rubisco with Rubisco Activase at 25.degree. C. There
were no heated conditions used. Column 6 of Table 3 represents the
amount of Rubisco that has been activated by Rubisco Activase when
the assay is conducted at 40.degree. C. as a percent of the amount
of Rubisco that has been activated by Rubisco Activase when the
assay is conducted at 25.degree. C.
[0107] Wild-type RCA maintained a Rubisco activation state of 0.5
at 40.degree. C. while Rubisco Activase derived polypeptides
183H12, 301C7 and 382D8 were able to maintain activation states of
0.62-0.72 under the same conditions (FIG. 1B). Relative to
reactions at 25.degree. C., the activation state of Rubisco
maintained by the thermostable variants at 40.degree. C. was in the
range of 78-98% versus 70% for the wild type enzyme. The protein
displaying the highest specific activity at either 25.degree. C. or
40.degree. C. was the best variant isolated in the first round,
183H12.
[0108] ATPase activity. Since Rubisco Activase is an ATPase (the
polypeptide contains the AAA.sup.+ domain) that requires ATP to
loosen the binding of Rubisco to sugar phosphates, the effect of
temperature on ATP hydrolysis by Rubisco Activase was tested. The
ATPase assay that monitored the intrinsic activity of the activase
complex regardless of its interaction with Rubisco is commonly used
for Rubisco Activase characterization. ATPase assay has been
performed as described by Salvucci ((1992) Arch. Biochem. Biophys.
298:688-696). Results are shown in columns 7-8 of Table 3. Column 7
of Table 3 represents the amount of hydrolyzed ATP that is present
after incubation of Rubisco Activase with ATP at 25.degree. C.
There were no heated conditions used. Column 8 of Table 3
represents the amount of hydrolyzed ATP that is present after
incubation with Rubisco Activase that had been heat treated at
40.degree. C. as a percent of the amount of hydrolyzed ATP that is
present after incubation with Rubisco Activase that had not been
heat treated.
[0109] FIG. 1C shows that the stability of Rubisco Activase derived
polypeptides 301C7 and 382D8 at 35.degree. C. and 40.degree. C. was
improved more than 10-fold compared to RCA1, whereas 183H12
exhibited 20% and 30% improvement at 25.degree. C. and 40.degree.
C., respectively.
Example 3
Complementation of Rubisco Activase Deletion Mutant
[0110] In order to express shuffled variants in homozygous
background for the deletion (.DELTA.rca/.DELTA.rca) (see, Li, et
al., (2001) Plant J. 27:235-242) the following complementation
cascade was developed: 1) Selection of heterozygous plants for the
deletion by HTP-PCR using single-leaf 96-well DNA extraction method
(Xin, et al., (2003) BioTechniques 34:820-826), with specific
primers for the wild-type and deleted alleles. 2) Transformation
with the gene of interest. 3) TO selection for antibiotic
resistance and PCR analysis for homozygosity. 4) Self pollination
of the resultant homozygous plants in order to obtain T1 transgenic
lines.
[0111] Immunoblot analysis of wild-type, heterozygous, and
homozygous plants (genetic background RCA/RCA, RCA/.DELTA.rca and
.DELTA.rca/.DELTA.rca respectively) revealed that the gene products
(long and short forms) were expressed at similar levels in
wild-type and heterozygous plants (FIG. 2A). The absence of the
short and long isoforms in plants homozygous for the deletion
confirmed that the mutation abrogates the expression of both RCA1
and RCA2. .DELTA.rca plants grown at ambient CO.sub.2 exhibited low
photosynthetic performance (Fq'/Fm' values) compared to wild-type
(0.185.+-.0.038 and 0.332.+-.0.033 respectively) (FIG. 2B) and
significant lower leaf area after 3 weeks on soil (2.93.+-.0.49 and
395.4.+-.8.75 mm.sup.2 respectively) (FIG. 2C). Two-month old
.DELTA.rca homozygotes were severely stunted and chlorotic by
comparison to wild-type plants (FIG. 2D).
[0112] Based on sequence analysis and mapping of the deleted
fragment, two sets of primers were designed: RCA primers (forward
5'-CAGACAATGTTGGCCTC-3' (SEQ ID NO: 23) and reverse
5'-ACGAGTAACGATGGTAGG-3' (SEQ ID NO: 24)) specific for the
wild-type allele that give 1.5 kb product, and rca primers (forward
5'-GTCTATACCTTGAGC-3' (SEQ ID NO: 25) and reverse
5'-TCAGTCATACTCGG-3' (SEQ ID NO: 26)) that give 1.5 kb product in
the deleted allele and 4.9 kb in the wild-type allele (FIG. 3A). In
order to amplify the 1.5 kb product with the rca primers but not
the 4.9 kb, the PCR amplification cycle was set to 1.5 min. Those
two sets of primers were utilized to characterize the genetic
background of the T1 plants. Since the transformation host was
heterozygous for deletion of the endogenous rca locus, the T1
plants expressing the Rubisco activase transgenes are a mixture of
wild-type (RCA/RCA) heterozygotes (RCA/.DELTA.rca) and homozygotes
(.DELTA.rca/.DELTA.rca). Transgenic lines expressing the shuffled
variant 183H12 in the different genetic backgrounds have been
identified using PCR screening (FIG. 3B) and immunoblot analysis
(FIG. 3C). Plants that express 183H12 in genetic backgrounds
containing at least one wild type allele (#13; wt, #12;
heterozygous for the deletion) have both the short and the long
forms of the protein. In line #2 (homozygous for the deletion) only
the short form was detected because the transgene was designed to
express only the short form of the protein.
Example 4
In Planta Characterization of Rubisco Activase Derived
Polypeptides
[0113] To determine the effect of improved Rubisco Activase under
normal and increased temperatures, the Arabidopsis Rubisco Activase
deletion mutant (.DELTA.rca) (see, Example 3). .DELTA.rca was
functionally complemented with wide type Rubisco Activase (SEQ ID
NO: 1), 1.sup.st round Rubisco Activase derived polypeptide 183H12
(SEQ ID NO: 7) and two 2.sup.nd round Rubisco Activase derived
polypeptides 382D8 (SEQ ID NO: 15) and 301C7 (SEQ ID NO: 19).
[0114] In order to express the wild type Rubisco Activase and the
Rubisco Activase derived polypeptides in transgenic Arabidopsis
plants (.DELTA.rca), the transgenes encoding the chloroplast
transit peptide and the coding region of rcal or the derived
polypeptides were cloned into pMAXY4384 that contains the Mirabilis
Mosaic Caulimovirus promoter (MMV) with a double enhancer domain
(Day and Maiti, (1999) Transgenics 3:61-70), the UBQ3 terminator
and the kanamycin resistance gene nptII. Heterozygous
Deleteagene.TM. RCA mutants were transformed by Agrobacterium
tumefaciens strain GV3101 using the floral dipping method (Clough,
et al., (1998) Plant J. 16:735-743). To confirm expression, protein
was extracted from plant tissue (2-3 g fresh weight) in liquid
N.sub.2 and 1 ml of extraction buffer (100 mM Tricine-KOH pH 8,
EDTA pH 8, 10 mM 2-mercaptoethanol, and Protease inhibitor cocktail
Set V). The crude extract was clarified by successive
centrifugation for 5 min at 3000 g, and 20 min at 12,000 g. Ten
micrograms of soluble protein extract was separated on 10%
SDS-polyacrylamide gels and transferred to a nitrocellulose
membrane (according to the instructions supplied by Invitrogen).
The blot was immunodecorated with the polyclonal antibodies raised
against the recombinant Arabidopsis RCA1 and the proteins were
detected using the Ap conjugated substrate kit (Bio-Rad).
[0115] As shown in FIG. 4A, wild-type plants (RCA/RCA) expressed
short and long isoforms of activase, whereas transgenic .DELTA.rca
lines complemented by the transgenes expressed only the 43 kDa
short isoform. Under 22.degree. C. culture conditions (plants grown
in 16 hour light (225 .mu.mol photons m.sup.-2 s.sup.-1)/8 hour
dark cycles), the transgenic lines exhibited similar growth rates
as the wild-type untransformed plants (FIG. 4B). Photosynthetic
performance (photosystem II operating efficiency Fq'/Fm') and
growth rates were analyzed using the chlorophyll a fluorescence
imaging system (Fluorlmager, Qubit Systems) as previously described
(Baker, et al., (2001) J. Exp. Bot. 52:615-621). The Fq'/Fm' values
of transgenic deletion lines expressing RCA1 or Rubisco Activase
derived polypeptides 183H12, 301C7 or 382D8 (.DELTA.rcaRCA1,
.DELTA.rca183H12, .DELTA.rca301C7 and .DELTA.rca382D8,
respectively) were similar to wild-type untransformed plants,
indicating that expression of the short form is sufficient for
functional complementation of .DELTA.rca under normal growth
conditions (FIG. 4C). Under these conditions the photosynthetic
activity measured by the portable infrared gas analyzer (L16400,
Li-Cor) under 150 .mu.mol photons m.sup.-2 s.sup.-1 and 350 pbar
CO.sub.2. of .DELTA.rcaRCA1-1 was similar to .DELTA.rca183H12-3,
.DELTA.rca301C7-3 and .DELTA.rca382D8-1 (FIG. 4D). Temporary
exposure to 30.degree. C. for 1 hr resulted in 12% decreased
photosynthesis in .DELTA.rcaRCA1-1. Conversely, lines .DELTA.rcal
83H12-3, .DELTA.rca301C7-3 and .DELTA.rca382D8-1 showed 16, 22 and
16% increased photosynthesis after 1 hr at 30.degree. C.
[0116] Since exposure of Arabidopsis plants to 30.degree. C. causes
minor induction of heat shock proteins (typically induced at
32.degree. C. and above) and minor effects on stomatal aperture
(Salvucci, et al., (2001) Plant Physiol. 127:1053-1064), growth
under prolonged heat treatment was conducted with Arabidopsis
plants expressing wild type or thermostable Rubisco Activase.
Four-week old transgenic lines exposed for two weeks to moderate
heat stress. Conditions of growth were 16 hours of light at 225
.mu.mol photons m.sup.-2 s.sup.-1 and 8 hours of dark. During the
light cycle, plants were grown at 22.degree. C. for 6 hours then
rapidly increased to 30.degree. C. (2.degree. C. per min) for 4
hours and then returned to 22.degree. C. for the completion of the
light cycle. During the dark cycle, plants remained at 22.degree.
C. Characterization of growth, biomass, and yield was performed as
previously described (Barth, et al., (2003) Heredity. 91:36-42).
The plants displayed normal phenotype and leaf color but varied in
size (FIG. 5A). .DELTA.rcaRCA1 (lines 1, 8 and 9) were stunted by
comparison to wild-type untransformed plants and to the .DELTA.rca
lines that express the Rubisco Activase derived polypeptides (FIG.
5B). Transgenic Arabidopsis expressing 183H12-3 that possesses the
highest in vitro specific activity were the largest plants. Lines
that expressed 301C7 and 382D8 were larger than .DELTA.rcaRCA1
lines but did not reach the leaf area levels of the 183H12 lines.
While .DELTA.rca lines expressing only the short form of the
wild-type gene (RCA1) were smaller than wild-type untransformed
lines (expressing both short and long forms), most transgenic lines
expressing the Rubisco Activase derived polypeptides exhibited
greater leaf area than wild-type untransformed plants (FIG. 5B),
and all lines expressing the Rubisco Activase derived polypeptides
were significantly larger (P=0.01) than the .DELTA.rca
transformants expressing RCA1.
[0117] Four-week old plants exposed to two weeks of moderate heat
stress also showed differences in rates of plant development. At
the end of the treatment period 74, 44 and 33% of .DELTA.rca183H12,
.DELTA.rca301C7 and .DELTA.rca382D8, respectively, had mature
inflorescence with open flowers, while 100% of untransformed
wild-type plants and 88% of .DELTA.rcaRCA1 lines had emerging
immature inflorescences with no open flowers (data not shown).
Additionally, 12% of the .DELTA.rcaRCA1 lines were in the
vegetative stage with no visible inflorescences. Under normal
growth conditions, Arabidopsis plants flower after four weeks.
Therefore, the relatively high percentage of .DELTA.rca183H12,
.DELTA.rca301C7 and .DELTA.rca382D8 lines showing normal
development is likely due to improved thermostability of RCA that
minimized the inhibition of photosynthesis and growth under
moderate heat stress conditions.
[0118] The best line from each variant was further analyzed for
photosynthetic activity during the moderate heat stress cycle
(after 2 hr at 30.degree. C.). Transgenic lines showed a CO.sub.2
fixation pattern that correlated with leaf area. Rates of CO.sub.2
fixation in lines .DELTA.rca183H12-3, .DELTA.rca301C7-3 and
.DELTA.rca382D8-1 were 30, 25 and 23% higher, respectively, than in
line .DELTA.rcaRCA1-1. These results demonstrated that Rubisco
activase is a limiting factor in photosynthesis under the
experimental conditions.
[0119] Mature plants (10 weeks old) exposed for 8 weeks to moderate
heat stress were similar in appearance. A slight positive effect on
plant height was detected in .DELTA.rca183H12-3, .DELTA.rca301C7-3
and .DELTA.rca382D8-1 lines (116, 121 and 119% respectively)
compared to .DELTA.rcaRCA1-1 (FIG. 5D). A dramatic difference was
observed in the number of siliques per plant, which was
130.8.+-.48.2, 84.3.+-.19.6 and 100.8.+-.26.9 for
.DELTA.rca183H12-3, .DELTA.rca301C7-3 and .DELTA.rca382D8-1
(respectively) compared to 40.2.+-.16.3 and 47.5.+-.15.8 for
.DELTA.rcaRCA1-1 and wild-type, respectively (FIG. 5E).
[0120] To confirm that the relatively enhanced formation of
siliques in transformants expressing improved RCA was not at the
expense of individual seed size, the weights of lots of 1000 seeds
were compared. As shown in FIG. 5F, .DELTA.rca183H12-3, and
.DELTA.rca382D8-1 produced slightly larger seeds (18 and 32%
respectively) than .DELTA.rcaRCA1-1, while the seed weight of
.DELTA.rca301C7-3 and wild-type plants was similar to that of
.DELTA.rcaRCA1-1.
[0121] To further analyze the effect of improved RCA on growth
under moderate temperature stress, T3 lines expressing the most
active clone at 25.degree. C. (in vitro), 183H12, were grown
continuously at 26.degree. C. under higher light intensity and
humidity than in the previous experiment. Conditions of growth were
16 hours of light at 300 .mu.mol photons m.sup.-2 s.sup.-1 and 8
hours of dark at 26.degree. C. and 85% humidity. Wild-type and
.DELTA.rcaRCA1 lines grown at 26.degree. C. produced slightly
decreased overall biomass and exhibited slow rates of plant
development than under normal growth conditions, whereas the
biomass and the developmental process of lines of .DELTA.rca183H12
was unchanged (not shown). In contrast, the number of siliques per
plant produced by .DELTA.rca plants grown at 26.degree. C. was
dramatically affected by the Rubisco Activase derived polypeptide
they expressed (FIG. 6A). .DELTA.rca183H12 lines possessed 50 to
100 more siliques per plant than .DELTA.rcaRCA1 lines and 40 to 80
more siliques per plant than wild-type plants. In addition, the
siliques of .DELTA.rca183H12 were larger than those of wild-type
plants and the .DELTA.rcaRCA1 lines and produced more seeds (FIG.
6B). Siliques from .DELTA.rca183H12 at 26.degree. C. exhibited a
similar phenotype to the wild-type grown under normal growth
conditions, but produced fewer seeds. Under normal growth
conditions a minor decrease in seed weight was observed in
.DELTA.rca lines expressing RCA1 and 183H12 compared to wild-type
plants (FIG. 6C; white bars). However, under continuous exposure to
26.degree. C., 50% to 150% greater seed weight was observed in
lines of .DELTA.rca183H12 than in either wild-type plants or lines
of .DELTA.rcaRCA1. In comparing seed weight for each line grown at
26.degree. C. to that of the same line grown at 22.degree. C.,
lines .DELTA.rca183H12-2, .DELTA.rca183H12-3 and
.DELTA.rca183H12-20 were strikingly less affected by the higher
growth temperature than the .DELTA.rcaRCA1 lines or the wild
type.
[0122] Since exposure to 26.degree. C. resulted in small siliques
containing few seeds of small seed-weight, seed viability was
analyzed using a germination test. Seeds from wild-type,
.DELTA.rcaRCA1-1 and .DELTA.rca183H12-3 were collected from plants
grown at normal growth conditions and 26.degree. C. and then
germinated at 22.degree. C. Seeds from .DELTA.rcaRCA1-1 and
.DELTA.rca183H12-3 lines of parents grown at 22.degree. C., showed
the same germination rate (86%), which was slightly lower than that
of wild-type plants (94%) (FIG. 6C). Complete inhibition of
germination (4%) was observed in .DELTA.rcaRCA1-1 seeds collected
from parents grown at 26.degree. C. and significant inhibition in
wild-type seeds (26%). Conversely, .DELTA.rca183H12-3 seeds
collected from parents grown at 26.degree. C. exhibited relatively
high germination rates of 70%.
[0123] Additionally plants were analyzed for photosynthesis rates,
growth rates and seed yield under the following growth
conditions:
[0124] Normal: Plants were grown under 16 hours light (225 .mu.mol
photons m.sup.-2 s.sup.-1) and 8 hour dark regime at 22.degree.
C.
[0125] Increased temperatures: Plants were grown under normal
growth conditions for two weeks and then transferred to the growth
chamber and grown under 16 hour light (225 .mu.mol photons m.sup.-2
s.sup.-1) and 8 hour dark regime. During the light cycle, the
temperature was set to 22.degree. C. for six hours and then rapidly
increased to 30.degree. C. (2.degree. C. per minute) for four
hours. After the heat treatment, the temperature was set back to
22.degree. C.
[0126] Continuous increased temperatures: Plants were grown under
normal growth conditions for two weeks and then transferred to the
growth room and grown under sixteen hours high light (300 .mu.mol
photons m.sup.-2 s.sup.-1) and eight hour dark regime. During the
light/dark cycle the temperature was set to 26.degree. C. and the
humidity to 80%.
[0127] The results of the in planta assays of Rubisco Activase
derived polypeptide activity at the conditions identified supra are
summarized below.
[0128] Growth rates. Leaf area was measured using the chlorophyll a
fluorescence imaging system (FluorImager, Qubit System Inc.). Leaf
area observed in untransformed wild type plants under the different
growth conditions was set to 100%. The data in Table 4 demonstrates
that plants expressing any of the three Rubisco Activase derived
polypeptides had increased growth rates under increased
temperatures as compared to either wild type plants or plants
expressing a transgenic wild type Rubisco Activase.
[0129] Photosynthesis rates. Plants were analyzed for CO.sub.2
fixation using the portable infrared gas analyzer (L16400, Li-Cor)
for 15 minutes. The light source was set to 225 .mu.mol photons
m.sup.-2 s.sup.-1 and the level of CO.sub.2 supplied to the leaf by
the built-in CO.sub.2 injection system was 350 .mu.mol m.sup.-2
s.sup.-1. The data in Table 5 shows that plants expressing any of
the three Rubisco Activase derived polypeptides had increased
photosynthetic rates under increased temperatures as compared to
either wild type plants or plants expressing a transgenic wild type
Rubisco Activase (see, column 3).
[0130] Seed yield. Seed weight (mg) was determined from mature
dried plants. Seed germination rate was determined by number of
plants germinated on MS plate supplemented with Kanamycin. The data
in Table 5 shows that plants expressing any of the three Rubisco
Activase derived polypeptides had increased seed yield under
increased temperatures as compared to either wild type plants or
plants expressing a transgenic wild type Rubisco Activase (see,
column 4). The increased seed yield was also present in plants
expressing any of the three Rubisco Activase derived polypeptides
under continuous increased temperature conditions (see, Table 6).
Germination rates of seeds was increased under continuous increased
temperature conditions in plants expressing the Rubisco Activase
derived polypeptide 183H12 (SEQ ID NO: 7) as compared to either
wild type plants or plants expressing a transgenic wild type
Rubisco Activase (see, Table 7).
TABLE-US-00001 TABLE 1 Codon Table Amino acid Codon Alanine Ala A
GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly
G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC
AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
TABLE-US-00002 TABLE 2 Effect of amino acids substitution on
Rubisco Activase activity and thermostability. Activity (3.sup.rd
tier) Thermo- Amino acid position .mu.mol CO.sub.2 min.sup.-1
mg.sup.-1 stability* Clone 42 130 131 168 257 274 293 310
25.degree. C. 40.degree. C. (%) Wild type M M M F V T R K 0.96 .+-.
0.04 0.634 .+-. 0.02 66 (SEQ ID NO: 1) 126H4 R 0.616 .+-. 0.04
0.596 .+-. 0.05 97 (SEQ ID NO: 3) 182B11 I 0.426 .+-. 0.03 0.359
.+-. 0.01 84 (SEQ ID NO: 5) 183H12 R 1.049 .+-. 0.02 0.857 .+-.
0.01 82 (SEQ ID NO: 7) 184B2 N 1.055 .+-. 0.03 0.889 .+-. 0.02 84
(SEQ ID NO: 9) 079H6 T I K 1.064 .+-. 0.01 0.940 .+-. 0.02 88 (SEQ
ID NO: 11) 214A4 T R 1.069 .+-. 0.06 0.986 .+-. 0.04 92 (SEQ ID NO:
13) 382D8 V I N 1.012 .+-. 0.05 0.91 .+-. 0.06 90 (SEQ ID NO: 15)
383A12 L I R N 0.891 .+-. 0.02 0.920 .+-. 0.02 103 (SEQ ID NO: 17)
301C7 L I N 0.905 .+-. 0.04 0.889 .+-. 0.01 98 (SEQ ID NO: 19)
301H3 I R N 0.971 .+-. 0.05 0.860 .+-. 0.05 89 (SEQ ID NO: 21)
*Percent of activated Rubisco at 40.degree. C. compared to amount
of activated Rubisco 25.degree. C.
TABLE-US-00003 TABLE 3 Relative activity (%) of Rubisco Activase
Derived Polypeptides. Activation of deactivated Rubisco activation
under Rubisco assay catalytic conditions assay ATPase assay T.S T.S
T.S T.S 45.degree. C./ 45 min at 40.degree. C./ 40.degree. C./
40.degree. C./ Clone 25.degree. C. 25.degree. C. 15 min at
25.degree. C. 25.degree. C. 25.degree. C. 25.degree. C. 25.degree.
C. Wild type 100 50 32 100 67 100 8 (SEQ ID NO: 1) 183H12 109 52 54
118 77 123 28 (SEQ ID NO: 7) 079H6 111 63 66 105 82 119 72 (SEQ ID
NO: 11) 214A4 111 58 65 101 74 124 38 (SEQ ID NO: 13) 382D8 105 86
85 98 93 112 113 (SEQ ID NO: 15) 383A12 93 92 88 96 97 93 92 (SEQ
ID NO: 17) 301C7 94 84 92 96 83 105 89 (SEQ ID NO: 19) 301H3 101 89
75 92 78 107 112 (SEQ ID NO: 21)
TABLE-US-00004 TABLE 4 Leaf area under normal and increased
temperature conditions. Leaf area (%)* Normal growth Increased
Clone Line ID conditions Temperatures Wild type Untransformed 100
.+-. 13 100 .+-. 30 Wild type RCA1-1 92 .+-. 26 62 .+-. 21 (SEQ ID
NO: 1) RCA1-8 106 .+-. 30 48 .+-. 16 RCA1-9 88 .+-. 27 45 .+-. 23
183H12 183H12-2 110 .+-. 25 131 .+-. 23 (SEQ ID NO: 7) 183H12-3 113
.+-. 17 142 .+-. 31 183H12-20 101 .+-. 30 138 .+-. 37 382D8 382D8-1
126 .+-. 23 151 .+-. 46 (SEQ ID NO: 15) 382D8-2 90 .+-. 31 81 .+-.
32 301C7 301C7-1 105 .+-. 30 101 .+-. 19 (SEQ ID NO: 19) 301C7-3
104 .+-. 30 124 .+-. 31 301C7-7 116 .+-. 23 115 .+-. 36 *The leaf
area of the Arabidopsis wild-type untransformed was set to
100%.
TABLE-US-00005 TABLE 5 Photosynthesis rates and seed yield under
normal and increased temperature conditions. Photosynthesis* Seed
Clone Line ID (.mu.mol CO.sub.2 m.sup.-2 s.sup.-1) yield.sup.# (%)
Wild type Untransformed 7.85 .+-. 0.36 72 .+-. 14 Wild type RCA1-1
7.25 .+-. 1.36 74 .+-. 12 (SEQ ID NO: 1) 183H12 183H12-3 9.41 .+-.
0.66 87 .+-. 8 (SEQ ID NO: 7) 382D8 382D8-1 8.9 .+-. 0.93 100 .+-.
11 (SEQ ID NO: 15) 301C7 301C7-3 9.03 .+-. 0.9 87 .+-. 16 (SEQ ID
NO: 19) *Net photosynthesis was monitored after 2 hours at
30.degree. C. .sup.#Percent of weight of 1000 seeds from plants
grown under increased temperatures as compared to weight of 1000
seeds from plants grown under normal conditions.
TABLE-US-00006 TABLE 6 Seed yield under normal and continuous
increased temperature conditions. 1000-seed weight (mg) Normal
growth Continuous increased Clone Line ID conditions temperatures
Wild type Untrans- 28.900 .+-. 4.140 9.484 .+-. 1.971 formed Wild
type RCA1-1 25.837 .+-. 2.721 8.501 .+-. 1.921 (SEQ ID NO: 1)
RCA1-8 21.880 .+-. 2.839 10.529 .+-. 0.830 RCA1-9 22.846 .+-. 1.714
9.861 .+-. 0.596 183H12 183H12-2 19.773 .+-. 2.112 15.858 .+-.
3.249 (SEQ ID NO:7) 183H12-3 25.934 .+-. 1.869 24.812 .+-. 1.376
183H12-20 21.730 .+-. 2.284 14.955 .+-. 2.996
TABLE-US-00007 TABLE 7 Germination rates of seeds harvested from
plants grown under normal and continuous increased temperature
conditions. Germinated seeds (%) Normal growth Continuous increased
Clone Line ID conditions temperatures Wild type Untrans- 94 .+-. 2
26 .+-. 6 formed Wild type RCA1-1 82 .+-. 6 4 .+-. 4 (SEQ ID NO: 1)
183H12 183H12-3 82 .+-. 2 70 .+-. 10 (SEQ ID NO: 7)
Sequence CWU 1
1
2611173DNAArabidopsis thaliana 1atggtgaaag aagacaaaca aaccgatgga
gacagatgga gaggtcttgc ctacgacact 60tctgatgatc aacaagacat caccagaggc
aagggtatgg ttgactctgt cttccaagct 120cctatgggaa ccggaactca
ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180cttaggcagt
acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg
240gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat
caaggttcca 300cttattttgg gtatatgggg aggcaaaggt caaggtaaat
ccttccagtg tgagcttgtc 360atggccaaga tgggtatcaa cccaatcatg
atgagtgctg gagagcttga gagtggaaac 420gcaggagaac ccgcaaagct
tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480aagggaaaga
tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt
540ggtactactc agtacactgt caacaaccag atggttaacg caacactcat
gaacattgct 600gataacccaa ccaacgtcca gctcccagga atgtacaaca
aggaagagaa cgcacgtgtc 660cccatcattt gcactggtaa cgatttctcc
accctatacg ctcctctcat ccgtgatgga 720cgtatggaga agttctactg
ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780atcttcagaa
ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct
840ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga
tgaagtgagg 900aagttcgttg agagccttgg agttgagaag atcggaaaga
ggctggttaa ctcaagggaa 960ggacctcccg tgttcgagca acccgagatg
acttatgaga agcttatgga atacggaaac 1020atgcttgtga tggaacaaga
gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080caggctgctt
tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa
1140acagaggaaa aggagcccag caagctcgag taa 11732390PRTArabidopsis
thaliana 2Met Val Lys Glu Asp Lys Gln Thr Asp Gly Asp Arg Trp Arg
Gly Leu1 5 10 15Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg
Gly Lys Gly 20 25 30Met Val Asp Ser Val Phe Gln Ala Pro Met Gly Thr
Gly Thr His His 35 40 45Ala Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln
Gly Leu Arg Gln Tyr 50 55 60Asn Leu Asp Asn Met Met Asp Gly Phe Tyr
Ile Ala Pro Ala Phe Met65 70 75 80Asp Lys Leu Val Val His Ile Thr
Lys Asn Phe Leu Thr Leu Pro Asn 85 90 95Ile Lys Val Pro Leu Ile Leu
Gly Ile Trp Gly Gly Lys Gly Gln Gly 100 105 110Lys Ser Phe Gln Cys
Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro 115 120 125Ile Met Met
Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro 130 135 140Ala
Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile Lys145 150
155 160Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly
Ala 165 170 175Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn
Gln Met Val 180 185 190Asn Ala Thr Leu Met Asn Ile Ala Asp Asn Pro
Thr Asn Val Gln Leu 195 200 205Pro Gly Met Tyr Asn Lys Glu Glu Asn
Ala Arg Val Pro Ile Ile Cys 210 215 220Thr Gly Asn Asp Phe Ser Thr
Leu Tyr Ala Pro Leu Ile Arg Asp Gly225 230 235 240Arg Met Glu Lys
Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly 245 250 255Val Cys
Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile 260 265
270Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe Phe Gly
275 280 285Ala Leu Arg Ala Arg Val Tyr Asp Asp Glu Val Arg Lys Phe
Val Glu 290 295 300Ser Leu Gly Val Glu Lys Ile Gly Lys Arg Leu Val
Asn Ser Arg Glu305 310 315 320Gly Pro Pro Val Phe Glu Gln Pro Glu
Met Thr Tyr Glu Lys Leu Met 325 330 335Glu Tyr Gly Asn Met Leu Val
Met Glu Gln Glu Asn Val Lys Arg Val 340 345 350Gln Leu Ala Glu Thr
Tyr Leu Ser Gln Ala Ala Leu Gly Asp Ala Asn 355 360 365Ala Asp Ala
Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys 370 375 380Glu
Pro Ser Lys Leu Glu385 39031173DNAArtificial SequenceBased on
Arabidopsis thaliana sequence 3atggtgaaag aagacaaaca aaccgatgga
gacagatgga gaggtcttgc ctacgacact 60tctgatgatc aacaagacat caccagaggc
aagggtatgg ttgactctgt cttccaagct 120cctatgggaa ccggaactca
ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180cttaggcagt
acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg
240gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat
caaggttcca 300cttattttgg gtatatgggg aggcaaaggt caaggtaaat
ccttccagtg tgagcttgtc 360atggccaaga tgggtatcaa cccaatcagg
atgagtgctg gagagcttga gagtggaaac 420gcaggagaac ccgcaaagct
tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480aagggaaaga
tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt
540ggtactactc agtacactgt caacaaccag atggttaacg caacactcat
gaacattgct 600gataacccaa ccaacgtcca gctcccagga atgtacaaca
aggaagagaa cgcacgtgtc 660cccatcattt gcactggtaa cgatttctcc
accctatacg ctcctctcat ccgtgatgga 720cgtatggaga agttctactg
ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780atcttcagaa
ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct
840ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga
tgaagtgagg 900aagttcgttg agagccttgg agttgagaag atcggaaaga
ggctggttaa ctcaagggaa 960ggacctcccg tgttcgagca acccgagatg
acttatgaga agcttatgga atacggaaac 1020atgcttgtga tggaacaaga
gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080caggctgctt
tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa
1140acagaggaaa aggagcccag caagctcgag taa 11734390PRTArtificial
SequenceBased on Arabidopsis thaliana sequence 4Met Val Lys Glu Asp
Lys Gln Thr Asp Gly Asp Arg Trp Arg Gly Leu1 5 10 15Ala Tyr Asp Thr
Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly 20 25 30Met Val Asp
Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His 35 40 45Ala Val
Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr 50 55 60Asn
Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met65 70 75
80Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn
85 90 95Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln
Gly 100 105 110Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly
Ile Asn Pro 115 120 125Ile Arg Met Ser Ala Gly Glu Leu Glu Ser Gly
Asn Ala Gly Glu Pro 130 135 140Ala Lys Leu Ile Arg Gln Arg Tyr Arg
Glu Ala Ala Asp Leu Ile Lys145 150 155 160Lys Gly Lys Met Cys Cys
Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala 165 170 175Gly Arg Met Gly
Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val 180 185 190Asn Ala
Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu 195 200
205Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys
210 215 220Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg
Asp Gly225 230 235 240Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg
Glu Asp Arg Ile Gly 245 250 255Val Cys Lys Gly Ile Phe Arg Thr Asp
Lys Ile Lys Asp Glu Asp Ile 260 265 270Val Thr Leu Val Asp Gln Phe
Pro Gly Gln Ser Ile Asp Phe Phe Gly 275 280 285Ala Leu Arg Ala Arg
Val Tyr Asp Asp Glu Val Arg Lys Phe Val Glu 290 295 300Ser Leu Gly
Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu305 310 315
320Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met
325 330 335Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys
Arg Val 340 345 350Gln Leu Ala Glu Thr Tyr Leu Ser Gln Ala Ala Leu
Gly Asp Ala Asn 355 360 365Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr
Gly Lys Thr Glu Glu Lys 370 375 380Glu Pro Ser Lys Leu Glu385
39051173DNAArtificial SequenceBased on Arabidopsis thaliana
sequence 5atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc
ctacgacact 60tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt
cttccaagct 120cctatgggaa ccggaactca ccacgctgtc cttagctcat
acgaatacgt tagccaaggc 180cttaggcagt acaacttgga caacatgatg
gatgggtttt acattgctcc tgctttcatg 240gacaagcttg ttgttcacat
caccaagaac ttcttgactc tgcctaacat caaggttcca 300cttattttgg
gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc
360atggccaaga tgggtatcaa cccaatcatg ataagtgctg gagagcttga
gagtggaaac 420gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg
aggcagctga cttgatcaag 480aagggaaaga tgtgttgtct cttcatcaac
gatcttgacg ctggtgcggg tcgtatgggt 540ggtactactc agtacactgt
caacaaccag atggttaacg caacactcat gaacattgct 600gataacccaa
ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc
660cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat
ccgtgatgga 720cgtatggaga agttctactg ggccccgacc cgtgaagacc
gtatcggtgt ctgcaagggt 780atcttcagaa ctgacaagat caaggacgaa
gacattgtca cacttgttga tcagttccct 840ggtcaatcta tcgatttctt
cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900aagttcgttg
agagccttgg agttgagaag atcggaaaga ggctggttaa ctcaagggaa
960ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga
atacggaaac 1020atgcttgtga tggaacaaga gaatgtcaag agagtccaac
ttgccgagac ctacctcagc 1080caggctgctt tgggagacgc aaacgctgac
gccatcggcc gcggaacttt ctacggtaaa 1140acagaggaaa aggagcccag
caagctcgag taa 11736390PRTArtificial SequenceBased on Arabidopsis
thaliana sequence 6Met Val Lys Glu Asp Lys Gln Thr Asp Gly Asp Arg
Trp Arg Gly Leu1 5 10 15Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile
Thr Arg Gly Lys Gly 20 25 30Met Val Asp Ser Val Phe Gln Ala Pro Met
Gly Thr Gly Thr His His 35 40 45Ala Val Leu Ser Ser Tyr Glu Tyr Val
Ser Gln Gly Leu Arg Gln Tyr 50 55 60Asn Leu Asp Asn Met Met Asp Gly
Phe Tyr Ile Ala Pro Ala Phe Met65 70 75 80Asp Lys Leu Val Val His
Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn 85 90 95Ile Lys Val Pro Leu
Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly 100 105 110Lys Ser Phe
Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro 115 120 125Ile
Met Ile Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro 130 135
140Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile
Lys145 150 155 160Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu
Asp Ala Gly Ala 165 170 175Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr
Val Asn Asn Gln Met Val 180 185 190Asn Ala Thr Leu Met Asn Ile Ala
Asp Asn Pro Thr Asn Val Gln Leu 195 200 205Pro Gly Met Tyr Asn Lys
Glu Glu Asn Ala Arg Val Pro Ile Ile Cys 210 215 220Thr Gly Asn Asp
Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly225 230 235 240Arg
Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly 245 250
255Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile
260 265 270Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe
Phe Gly 275 280 285Ala Leu Arg Ala Arg Val Tyr Asp Asp Glu Val Arg
Lys Phe Val Glu 290 295 300Ser Leu Gly Val Glu Lys Ile Gly Lys Arg
Leu Val Asn Ser Arg Glu305 310 315 320Gly Pro Pro Val Phe Glu Gln
Pro Glu Met Thr Tyr Glu Lys Leu Met 325 330 335Glu Tyr Gly Asn Met
Leu Val Met Glu Gln Glu Asn Val Lys Arg Val 340 345 350Gln Leu Ala
Glu Thr Tyr Leu Ser Gln Ala Ala Leu Gly Asp Ala Asn 355 360 365Ala
Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys 370 375
380Glu Pro Ser Lys Leu Glu385 39071173DNAArtificial SequenceBased
on Arabidopsis thaliana sequence 7atggtgaaag aagacaaaca aaccgatgga
gacagatgga gaggtcttgc ctacgacact 60tctgatgatc aacaagacat caccagaggc
aagggtatgg ttgactctgt cttccaagct 120cctatgggaa ccggaactca
ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180cttaggcagt
acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg
240gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat
caaggttcca 300cttattttgg gtatatgggg aggcaaaggt caaggtaaat
ccttccagtg tgagcttgtc 360atggccaaga tgggtatcaa cccaatcatg
atgagtgctg gagagcttga gagtggaaac 420gcaggagaac ccgcaaagct
tatccgtcag aggtaccgtg aggcagctga tttgatcaag 480aagggaaaga
tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt
540ggtactactc agtacactgt caacaaccag atggttaacg caacactcat
gaacattgct 600gataacccaa ccaacgtcca gctcccagga atgtacaaca
aggaagagaa cgcacgtgtc 660cccatcattt gcactggtaa cgatttctcc
accctatacg ctcctctcat ccgtgatgga 720cgtatggaga agttctactg
ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780atcttcagaa
ctgacaagat caaggacgaa gacattgtca gacttgttga tcagttccct
840ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga
tgaagtgagg 900aagttcgttg agagccttgg agttgagaag atcggaaaga
ggctggttaa ctcaagggaa 960ggacctcccg tgttcgagca acccgagatg
acttatgaga agcttatgga atacggaaac 1020atgcttgtga tggaacaaga
gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080caggctgctc
tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa
1140acagaggaaa aggagcccag caagctcgag taa 11738390PRTArtificial
SequenceBased on Arabidopsis thaliana sequence 8Met Val Lys Glu Asp
Lys Gln Thr Asp Gly Asp Arg Trp Arg Gly Leu1 5 10 15Ala Tyr Asp Thr
Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly 20 25 30Met Val Asp
Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His 35 40 45Ala Val
Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr 50 55 60Asn
Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met65 70 75
80Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn
85 90 95Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln
Gly 100 105 110Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly
Ile Asn Pro 115 120 125Ile Met Met Ser Ala Gly Glu Leu Glu Ser Gly
Asn Ala Gly Glu Pro 130 135 140Ala Lys Leu Ile Arg Gln Arg Tyr Arg
Glu Ala Ala Asp Leu Ile Lys145 150 155 160Lys Gly Lys Met Cys Cys
Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala 165 170 175Gly Arg Met Gly
Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val 180 185 190Asn Ala
Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu 195 200
205Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys
210 215 220Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg
Asp Gly225 230 235 240Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg
Glu Asp Arg Ile Gly 245 250 255Val Cys Lys Gly Ile Phe Arg Thr Asp
Lys Ile Lys Asp Glu Asp Ile 260 265 270Val Arg Leu Val Asp Gln Phe
Pro Gly Gln Ser Ile Asp Phe Phe Gly 275 280 285Ala Leu Arg Ala Arg
Val Tyr Asp Asp Glu Val Arg Lys Phe Val Glu 290 295 300Ser Leu Gly
Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu305 310 315
320Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met
325 330 335Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys
Arg Val 340 345 350Gln Leu Ala Glu Thr Tyr Leu Ser Gln Ala Ala Leu
Gly Asp Ala Asn 355 360 365Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr
Gly Lys Thr Glu Glu Lys 370 375 380Glu Pro Ser Lys Leu Glu385
39091173DNAArtificial SequenceBased on Arabidopsis thaliana
sequence 9atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc
ctacgacact 60tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt
cttccaagct 120cctatgggaa ccggaactca ccacgctgtc cttagctcat
acgaatacgt tagccaaggc 180cttaggcagt acaacttgga caacatgatg
gatgggtttt acattgctcc tgctttcatg 240gacaagcttg ttgttcacat
caccaagaac ttcttgactc tgcctaacat caaggttcca 300cttattttgg
gtatatgggg aggcaaaggt caaggtaaat ccttccagtg
tgagcttgtc 360atggccaaga tgggtatcaa cccaatcatg atgagtgctg
gagagcttga gagtggaaac 420gcaggagaac ccgcaaagct tatccgtcag
aggtaccgtg aggcagctga cttgatcaag 480aagggaaaga tgtgttgtct
cttcatcaac gatcttgacg ctggtgcggg tcgtatggga 540ggtactactc
agtacactgt caacaaccag atggttaacg caacactcat gaacattgct
600gataacccaa ccaacgtcca gctcccagga atgtacaaca aggaagagaa
cgcacgtgtc 660cccatcattt gcactggtaa cgatttctcc accctatacg
ctcctctcat ccgtgatgga 720cgtatggaga agttctactg ggccccgacc
cgtgaagacc gtatcggtgt ctgcaagggt 780atcttcagaa ctgacaagat
caaggacgaa gacattgtca cacttgttga tcagttccct 840ggtcaatcta
tcgatttctt cggtgctttg agggcgagag tgtacgatga tgaagtgagg
900aagttcgttg agagccttgg agttgagaat atcggaaaga ggctggttaa
ctcaagggaa 960ggacctcccg tgttcgagca acccgagatg acttatgaga
agcttatgga atacggaaac 1020atgcttgtga tggaacaaga gaatgtcaag
agagtccaac ttgccgagac ctacctcagc 1080caggctgctt tgggagacgc
aaacgctgac gccatcggcc gcggaacttt ctacggtaaa 1140acagaggaaa
aggagcccag caagctcgag taa 117310390PRTArtificial SequenceBased on
Arabidopsis thaliana sequence 10Met Val Lys Glu Asp Lys Gln Thr Asp
Gly Asp Arg Trp Arg Gly Leu1 5 10 15Ala Tyr Asp Thr Ser Asp Asp Gln
Gln Asp Ile Thr Arg Gly Lys Gly 20 25 30Met Val Asp Ser Val Phe Gln
Ala Pro Met Gly Thr Gly Thr His His 35 40 45Ala Val Leu Ser Ser Tyr
Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr 50 55 60Asn Leu Asp Asn Met
Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met65 70 75 80Asp Lys Leu
Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn 85 90 95Ile Lys
Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly 100 105
110Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro
115 120 125Ile Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly
Glu Pro 130 135 140Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala
Asp Leu Ile Lys145 150 155 160Lys Gly Lys Met Cys Cys Leu Phe Ile
Asn Asp Leu Asp Ala Gly Ala 165 170 175Gly Arg Met Gly Gly Thr Thr
Gln Tyr Thr Val Asn Asn Gln Met Val 180 185 190Asn Ala Thr Leu Met
Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu 195 200 205Pro Gly Met
Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys 210 215 220Thr
Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly225 230
235 240Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile
Gly 245 250 255Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp
Glu Asp Ile 260 265 270Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser
Ile Asp Phe Phe Gly 275 280 285Ala Leu Arg Ala Arg Val Tyr Asp Asp
Glu Val Arg Lys Phe Val Glu 290 295 300Ser Leu Gly Val Glu Asn Ile
Gly Lys Arg Leu Val Asn Ser Arg Glu305 310 315 320Gly Pro Pro Val
Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met 325 330 335Glu Tyr
Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys Arg Val 340 345
350Gln Leu Ala Glu Thr Tyr Leu Ser Gln Ala Ala Leu Gly Asp Ala Asn
355 360 365Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu
Glu Lys 370 375 380Glu Pro Ser Lys Leu Glu385
390111173DNAArtificial SequenceBased on Arabidopsis thaliana
sequence 11atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc
ctacgacact 60tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt
cttccaagct 120cctacgggaa ccggaactca ccacgctgtc cttagctcat
acgaatacgt tagccaaggc 180cttaggcagt acaacttgga caacatgatg
gatgggtttt acattgctcc tgctttcatg 240gacaagcttg ttgttcacat
caccaagaac ttcttgactc tgcctaacat caaggttcca 300cttattttgg
gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc
360atggccaaga tgggtatcaa cccaatcatg ataagtgctg gagagcttga
gagtggaaac 420gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg
aggcagctga cttgatcaag 480aagggaaaga tgtgttgtct cttcatcaac
gatcttgacg ctggtgcggg tcgtatgggt 540ggtactactc agtacactgt
caacaaccag atggttaacg caacactcat gaacattgct 600gataacccaa
ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc
660cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat
ccgtgatgga 720cgtatggaga agttctactg ggccccgacc cgtgaagacc
gtatcggtgt ctgcaagggt 780atcttcagaa ctgacaagat caaggacgaa
gacattgtca cacttgttga tcagttccct 840ggtcaatcta tcgatttctt
cggtgctttg agggcgaaag tgtacgatga tgaagtgagg 900aagttcgttg
agagccttgg agttgagaag atcggaaaga ggctggttaa ctcaagggaa
960ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga
atacggaaac 1020atgctcgtga tggaacaaga gaatgtcaag agagtccaac
ttgccgagac ctacctcagc 1080caggctgctt tgggagacgc aaacgctgac
gccatcggcc gcggaacttt ctacggtaaa 1140acagaggaaa aggagcccag
caagctcgag taa 117312390PRTArtificial SequenceBased on Arabidopsis
thaliana sequence 12Met Val Lys Glu Asp Lys Gln Thr Asp Gly Asp Arg
Trp Arg Gly Leu1 5 10 15Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile
Thr Arg Gly Lys Gly 20 25 30Met Val Asp Ser Val Phe Gln Ala Pro Thr
Gly Thr Gly Thr His His 35 40 45Ala Val Leu Ser Ser Tyr Glu Tyr Val
Ser Gln Gly Leu Arg Gln Tyr 50 55 60Asn Leu Asp Asn Met Met Asp Gly
Phe Tyr Ile Ala Pro Ala Phe Met65 70 75 80Asp Lys Leu Val Val His
Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn 85 90 95Ile Lys Val Pro Leu
Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly 100 105 110Lys Ser Phe
Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro 115 120 125Ile
Met Ile Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro 130 135
140Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile
Lys145 150 155 160Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu
Asp Ala Gly Ala 165 170 175Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr
Val Asn Asn Gln Met Val 180 185 190Asn Ala Thr Leu Met Asn Ile Ala
Asp Asn Pro Thr Asn Val Gln Leu 195 200 205Pro Gly Met Tyr Asn Lys
Glu Glu Asn Ala Arg Val Pro Ile Ile Cys 210 215 220Thr Gly Asn Asp
Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly225 230 235 240Arg
Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly 245 250
255Val Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile
260 265 270Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe
Phe Gly 275 280 285Ala Leu Arg Ala Lys Val Tyr Asp Asp Glu Val Arg
Lys Phe Val Glu 290 295 300Ser Leu Gly Val Glu Lys Ile Gly Lys Arg
Leu Val Asn Ser Arg Glu305 310 315 320Gly Pro Pro Val Phe Glu Gln
Pro Glu Met Thr Tyr Glu Lys Leu Met 325 330 335Glu Tyr Gly Asn Met
Leu Val Met Glu Gln Glu Asn Val Lys Arg Val 340 345 350Gln Leu Ala
Glu Thr Tyr Leu Ser Gln Ala Ala Leu Gly Asp Ala Asn 355 360 365Ala
Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys 370 375
380Glu Pro Ser Lys Leu Glu385 390131173DNAArtificial SequenceBased
on Arabidopsis thaliana sequence 13atggtgaaag aagacaaaca aaccgatgga
gacagatgga gaggtcttgc ctacgacact 60tctgatgatc aacaagacat caccagaggc
aagggtatgg ttgactctgt cttccaagct 120cctacgggaa ccggaactca
ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180cttaggcagt
acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg
240gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat
caaggttcca 300cttattttgg gtatatgggg aggcaaaggt caaggtaaat
ccttccagtg tgagcttgtc 360atggccaaga tgggtatcaa cccaatcatg
atgagtgctg gagagcttga gagtggaaac 420gcaggagaac ccgcaaagct
tatccgtcag aggtaccgtg aggcagctga tttgatcaag 480aagggaaaga
tgtgttgtct cttcatcaac gatcttgacg ctggtgcggg tcgtatgggt
540ggtactactc agtacactgt caacaaccag atggttaacg caacactcat
gaacattgct 600gataacccaa ccaacgtcca gctcccagga atgtacaaca
aggaagagaa cgcacgtgtc 660cccatcattt gcactggtaa cgatttctcc
accctatacg ctcctctcat ccgtgatgga 720cgtatggaga agttctactg
ggccccgacc cgtgaagacc gtatcggtgt ctgcaagggt 780atcttcagaa
ctgacaagat caaggacgaa gacattgtca gacttgttga tcagttccct
840ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga
tgaagtgagg 900aagttcgttg agagccttgg agttgagaag atcggaaaga
ggctggttaa ctcaagggaa 960ggacctcccg tgttcgagca acccgagatg
acttatgaga agcttatgga atacggaaac 1020atgctcgtga tggaacaaga
gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080caggctgctt
tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa
1140acagaggaaa aggagcccag caagctcgag taa 117314390PRTArtificial
SequenceBased on Arabidopsis thaliana sequence 14Met Val Lys Glu
Asp Lys Gln Thr Asp Gly Asp Arg Trp Arg Gly Leu1 5 10 15Ala Tyr Asp
Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly 20 25 30Met Val
Asp Ser Val Phe Gln Ala Pro Thr Gly Thr Gly Thr His His 35 40 45Ala
Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr 50 55
60Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met65
70 75 80Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro
Asn 85 90 95Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly
Gln Gly 100 105 110Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met
Gly Ile Asn Pro 115 120 125Ile Met Met Ser Ala Gly Glu Leu Glu Ser
Gly Asn Ala Gly Glu Pro 130 135 140Ala Lys Leu Ile Arg Gln Arg Tyr
Arg Glu Ala Ala Asp Leu Ile Lys145 150 155 160Lys Gly Lys Met Cys
Cys Leu Phe Ile Asn Asp Leu Asp Ala Gly Ala 165 170 175Gly Arg Met
Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val 180 185 190Asn
Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu 195 200
205Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys
210 215 220Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg
Asp Gly225 230 235 240Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg
Glu Asp Arg Ile Gly 245 250 255Val Cys Lys Gly Ile Phe Arg Thr Asp
Lys Ile Lys Asp Glu Asp Ile 260 265 270Val Arg Leu Val Asp Gln Phe
Pro Gly Gln Ser Ile Asp Phe Phe Gly 275 280 285Ala Leu Arg Ala Arg
Val Tyr Asp Asp Glu Val Arg Lys Phe Val Glu 290 295 300Ser Leu Gly
Val Glu Lys Ile Gly Lys Arg Leu Val Asn Ser Arg Glu305 310 315
320Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met
325 330 335Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys
Arg Val 340 345 350Gln Leu Ala Glu Thr Tyr Leu Ser Gln Ala Ala Leu
Gly Asp Ala Asn 355 360 365Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr
Gly Lys Thr Glu Glu Lys 370 375 380Glu Pro Ser Lys Leu Glu385
390151173DNAArtificial SequenceBased on Arabidopsis thaliana
sequence 15atggtgaaag aagacaaaca aaccgatgga gacagatgga gaggtcttgc
ctacgacact 60tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt
cttccaagct 120cctatgggaa ccggaactca ccacgctgtc cttagctcat
acgaatacgt tagccaaggc 180cttaggcagt acaacttgga caacatgatg
gatgggtttt acattgctcc tgctttcatg 240gacaagcttg ttgttcacat
caccaagaac ttcttgactc tgcctaacat caaggttcca 300cttattttgg
gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc
360atggccaaga tgggtatcaa cccaatcatg gtgagtgctg gagagcttga
gagtggaaac 420gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg
aggcagctga tttgatcaag 480aagggaaaga tgtgttgtct cttcatcaac
gatcttgacg ctggtgcggg tcgtatgggt 540ggtactactc agtacactgt
caacaaccag atggttaacg caacactcat gaacattgct 600gataacccaa
ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc
660cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat
ccgtgatgga 720cgtatggaga agttctactg ggccccgacc cgtgaagacc
gtatcggtat atgcaagggt 780atcttcagaa ctgacaagat caaggacgaa
gacattgtca cacttgttga tcagttccct 840ggtcaatcta tcgatttctt
cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900aagttcgttg
agagccttgg agttgagaat atcggaaaga ggctggttaa ctcaagggaa
960ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga
atacggaaac 1020atgcttgtga tggaacaaga gaatgtcaag agagtccaac
ttgccgagac ctacctcagc 1080caggctgctt tgggagacgc aaacgctgac
gccatcggcc gcggaacttt ctacggtaaa 1140acagaggaaa aggagcccag
caagctcgag taa 117316390PRTArtificial SequenceBased on Arabidopsis
thaliana sequence 16Met Val Lys Glu Asp Lys Gln Thr Asp Gly Asp Arg
Trp Arg Gly Leu1 5 10 15Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile
Thr Arg Gly Lys Gly 20 25 30Met Val Asp Ser Val Phe Gln Ala Pro Met
Gly Thr Gly Thr His His 35 40 45Ala Val Leu Ser Ser Tyr Glu Tyr Val
Ser Gln Gly Leu Arg Gln Tyr 50 55 60Asn Leu Asp Asn Met Met Asp Gly
Phe Tyr Ile Ala Pro Ala Phe Met65 70 75 80Asp Lys Leu Val Val His
Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn 85 90 95Ile Lys Val Pro Leu
Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly 100 105 110Lys Ser Phe
Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro 115 120 125Ile
Met Val Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro 130 135
140Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile
Lys145 150 155 160Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu
Asp Ala Gly Ala 165 170 175Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr
Val Asn Asn Gln Met Val 180 185 190Asn Ala Thr Leu Met Asn Ile Ala
Asp Asn Pro Thr Asn Val Gln Leu 195 200 205Pro Gly Met Tyr Asn Lys
Glu Glu Asn Ala Arg Val Pro Ile Ile Cys 210 215 220Thr Gly Asn Asp
Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly225 230 235 240Arg
Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly 245 250
255Ile Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile
260 265 270Val Thr Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe
Phe Gly 275 280 285Ala Leu Arg Ala Arg Val Tyr Asp Asp Glu Val Arg
Lys Phe Val Glu 290 295 300Ser Leu Gly Val Glu Asn Ile Gly Lys Arg
Leu Val Asn Ser Arg Glu305 310 315 320Gly Pro Pro Val Phe Glu Gln
Pro Glu Met Thr Tyr Glu Lys Leu Met 325 330 335Glu Tyr Gly Asn Met
Leu Val Met Glu Gln Glu Asn Val Lys Arg Val 340 345 350Gln Leu Ala
Glu Thr Tyr Leu Ser Gln Ala Ala Leu Gly Asp Ala Asn 355 360 365Ala
Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys 370 375
380Glu Pro Ser Lys Leu Glu385 390171173DNAArtificial SequenceBased
on Arabidopsis thaliana sequence 17atggtgaaag aagacaaaca aaccgatgga
gacagatgga gaggtcttgc ctacgacact 60tctgatgatc aacaagacat caccagaggc
aagggtatgg ttgactctgt cttccaagct 120cctatgggaa ccggaactca
ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180cttaggcagt
acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg
240gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat
caaggttcca 300cttattttgg gtatatgggg aggcaaaggt caaggtaaat
ccttccagtg tgagcttgtc 360atggccaaga tgggtatcaa cccaatcatg
atgagtgctg gagagcttga gagtggaaac 420gcaggagaac ccgcaaagct
tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480aagggaaaga
tgtgttgtct cctcatcaac gatcttgacg ctggtgcggg tcgtatgggt
540ggtactactc agtacactgt caacaaccag atggttaacg caacactcat
gaacattgct 600gataacccaa ccaacgtcca gctcccagga atgtacaaca
aggaagagaa cgcacgtgtc
660cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat
ccgtgatgga 720cgtatggaga agttctactg ggccccgacc cgtgaagacc
gtatcggtat atgcaagggt 780atcttcagaa ctgacaagat caaggacgaa
gacattgtca gacttgttga tcagttccct 840ggtcaatcta tcgatttctt
cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900aagttcgttg
agagccttgg agttgagaat atcggaaaga ggctggttaa ctcaagggaa
960ggacctcccg tgttcgagca acccgagatg acttatgaga agcttatgga
atacggaaac 1020atgcttgtga tggaacaaga gaatgtcaag agagtccaac
ttgccgagac ctacctcagc 1080caggctgctc tgggagacgc aaacgctgac
gccatcggcc gcggaacttt ctacggtaaa 1140acagaggaaa aggagcccag
caagctcgag taa 117318390PRTArtificial SequenceBased on Arabidopsis
thaliana sequence 18Met Val Lys Glu Asp Lys Gln Thr Asp Gly Asp Arg
Trp Arg Gly Leu1 5 10 15Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile
Thr Arg Gly Lys Gly 20 25 30Met Val Asp Ser Val Phe Gln Ala Pro Met
Gly Thr Gly Thr His His 35 40 45Ala Val Leu Ser Ser Tyr Glu Tyr Val
Ser Gln Gly Leu Arg Gln Tyr 50 55 60Asn Leu Asp Asn Met Met Asp Gly
Phe Tyr Ile Ala Pro Ala Phe Met65 70 75 80Asp Lys Leu Val Val His
Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn 85 90 95Ile Lys Val Pro Leu
Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly 100 105 110Lys Ser Phe
Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro 115 120 125Ile
Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro 130 135
140Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile
Lys145 150 155 160Lys Gly Lys Met Cys Cys Leu Leu Ile Asn Asp Leu
Asp Ala Gly Ala 165 170 175Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr
Val Asn Asn Gln Met Val 180 185 190Asn Ala Thr Leu Met Asn Ile Ala
Asp Asn Pro Thr Asn Val Gln Leu 195 200 205Pro Gly Met Tyr Asn Lys
Glu Glu Asn Ala Arg Val Pro Ile Ile Cys 210 215 220Thr Gly Asn Asp
Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly225 230 235 240Arg
Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly 245 250
255Ile Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile
260 265 270Val Arg Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe
Phe Gly 275 280 285Ala Leu Arg Ala Arg Val Tyr Asp Asp Glu Val Arg
Lys Phe Val Glu 290 295 300Ser Leu Gly Val Glu Asn Ile Gly Lys Arg
Leu Val Asn Ser Arg Glu305 310 315 320Gly Pro Pro Val Phe Glu Gln
Pro Glu Met Thr Tyr Glu Lys Leu Met 325 330 335Glu Tyr Gly Asn Met
Leu Val Met Glu Gln Glu Asn Val Lys Arg Val 340 345 350Gln Leu Ala
Glu Thr Tyr Leu Ser Gln Ala Ala Leu Gly Asp Ala Asn 355 360 365Ala
Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys 370 375
380Glu Pro Ser Lys Leu Glu385 390191173DNAArtificial SequenceBased
on Arabidopsis thaliana sequence 19atggtgaaag aagacaaaca aaccgacgga
gacagatgga gaggtcttgc ctacgacact 60tctgatgatc aacaagacat caccagaggc
aagggtatgg ttgactctgt cttccaagct 120cctatgggaa ccggaactca
ccacgctgtc cttagctcat acgaatacgt tagccaaggc 180cttaggcagt
acaacttgga caacatgatg gatgggtttt acattgctcc tgctttcatg
240gacaagcttg ttgttcacat caccaagaac ttcttgactc tgcctaacat
caaggttcca 300cttattttgg gtatatgggg aggcaaaggt caaggtaaat
ccttccagtg tgagcttgtc 360atggccaaga tgggtatcaa cccaatcatg
atgagtgctg gagagcttga gagtggaaac 420gcaggagaac ccgcaaagct
tatccgtcag aggtaccgtg aggcagctga cttgatcaag 480aagggaaaga
tgtgttgtct cctcatcaac gatcttgacg ctggtgcggg tcgtatgggt
540ggtactactc agtacactgt caacaaccag atggttaacg caacactcat
gaacattgct 600gataacccaa ccaacgtcca gctcccagga atgtacaaca
aggaagagaa cgcacgtgtc 660cccatcattt gcactggtaa cgatttctcc
accctatacg ctcctctcat ccgtgatgga 720cgtatggaga agttctactg
ggccccgacc cgtgaagacc gtatcggtat atgcaagggt 780atcttcagaa
ctgacaagat caaggacgaa gacattgtca cacttgttga tcagttccct
840ggtcaatcta tcgatttctt cggtgctttg agggcgagag tgtacgatga
tgaagtgagg 900aagttcgttg agagccttgg agttgagaat atcggaaaga
ggctggttaa ctcaagggaa 960ggacctcccg tgttcgagca acccgagatg
acttatgaga agcttatgga atacggaaac 1020atgctcgtga tggaacaaga
gaatgtcaag agagtccaac ttgccgagac ctacctcagc 1080caggctgctt
tgggagacgc aaacgctgac gccatcggcc gcggaacttt ctacggtaaa
1140acagaggaaa aggagcccag caagctcgag taa 117320390PRTArtificial
SequenceBased on Arabidopsis thaliana sequence 20Met Val Lys Glu
Asp Lys Gln Thr Asp Gly Asp Arg Trp Arg Gly Leu1 5 10 15Ala Tyr Asp
Thr Ser Asp Asp Gln Gln Asp Ile Thr Arg Gly Lys Gly 20 25 30Met Val
Asp Ser Val Phe Gln Ala Pro Met Gly Thr Gly Thr His His 35 40 45Ala
Val Leu Ser Ser Tyr Glu Tyr Val Ser Gln Gly Leu Arg Gln Tyr 50 55
60Asn Leu Asp Asn Met Met Asp Gly Phe Tyr Ile Ala Pro Ala Phe Met65
70 75 80Asp Lys Leu Val Val His Ile Thr Lys Asn Phe Leu Thr Leu Pro
Asn 85 90 95Ile Lys Val Pro Leu Ile Leu Gly Ile Trp Gly Gly Lys Gly
Gln Gly 100 105 110Lys Ser Phe Gln Cys Glu Leu Val Met Ala Lys Met
Gly Ile Asn Pro 115 120 125Ile Met Met Ser Ala Gly Glu Leu Glu Ser
Gly Asn Ala Gly Glu Pro 130 135 140Ala Lys Leu Ile Arg Gln Arg Tyr
Arg Glu Ala Ala Asp Leu Ile Lys145 150 155 160Lys Gly Lys Met Cys
Cys Leu Leu Ile Asn Asp Leu Asp Ala Gly Ala 165 170 175Gly Arg Met
Gly Gly Thr Thr Gln Tyr Thr Val Asn Asn Gln Met Val 180 185 190Asn
Ala Thr Leu Met Asn Ile Ala Asp Asn Pro Thr Asn Val Gln Leu 195 200
205Pro Gly Met Tyr Asn Lys Glu Glu Asn Ala Arg Val Pro Ile Ile Cys
210 215 220Thr Gly Asn Asp Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg
Asp Gly225 230 235 240Arg Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg
Glu Asp Arg Ile Gly 245 250 255Ile Cys Lys Gly Ile Phe Arg Thr Asp
Lys Ile Lys Asp Glu Asp Ile 260 265 270Val Thr Leu Val Asp Gln Phe
Pro Gly Gln Ser Ile Asp Phe Phe Gly 275 280 285Ala Leu Arg Ala Arg
Val Tyr Asp Asp Glu Val Arg Lys Phe Val Glu 290 295 300Ser Leu Gly
Val Glu Asn Ile Gly Lys Arg Leu Val Asn Ser Arg Glu305 310 315
320Gly Pro Pro Val Phe Glu Gln Pro Glu Met Thr Tyr Glu Lys Leu Met
325 330 335Glu Tyr Gly Asn Met Leu Val Met Glu Gln Glu Asn Val Lys
Arg Val 340 345 350Gln Leu Ala Glu Thr Tyr Leu Ser Gln Ala Ala Leu
Gly Asp Ala Asn 355 360 365Ala Asp Ala Ile Gly Arg Gly Thr Phe Tyr
Gly Lys Thr Glu Glu Lys 370 375 380Glu Pro Ser Lys Leu Glu385
390211173DNAArtificial SequenceBased on Arabidopsis thaliana
sequence 21atggtgaaag aagacaaaca aaccgacgga gacagatgga gaggtcttgc
ctacgacact 60tctgatgatc aacaagacat caccagaggc aagggtatgg ttgactctgt
cttccaagct 120cctatgggaa ccggaactca ccacgctgtc cttagctcat
acgaatacgt tagccaaggc 180cttaggcagt acaacttgga caacatgatg
gatgggtttt acattgctcc tgctttcatg 240gacaagcttg ttgttcacat
caccaagaac ttcttgactc tgcctaacat caaggttcca 300cttattttgg
gtatatgggg aggcaaaggt caaggtaaat ccttccagtg tgagcttgtc
360atggccaaga tgggtatcaa cccaatcatg atgagtgctg gagagcttga
gagtggaaac 420gcaggagaac ccgcaaagct tatccgtcag aggtaccgtg
aggcagctga cttgatcaag 480aagggaaaga tgtgttgtct cttcatcaac
gatcttgacg ctggtgcggg tcgtatgggt 540ggtactactc agtacactgt
caacaaccag atggttaacg caacactcat gaacattgct 600gataacccaa
ccaacgtcca gctcccagga atgtacaaca aggaagagaa cgcacgtgtc
660cccatcattt gcactggtaa cgatttctcc accctatacg ctcctctcat
ccgtgatgga 720cgtatggaga agttctactg ggccccgacc cgtgaagacc
gtatcggtat atgcaagggt 780atcttcagaa ctgacaagat caaggacgaa
gacattgtca gacttgttga tcagttccct 840ggtcaatcta tcgatttctt
cggtgctttg agggcgagag tgtacgatga tgaagtgagg 900aagttcgttg
agagccttgg agttgagaat atcggaaaga ggctggttaa ctcaagggaa
960ggaccccccg tgttcgagca acccgagatg acttatgaga agcttatgga
atacggaaac 1020atgcttgtga tggaacaaga gaatgtcaag agagtccaac
ttgccgagac ctacctcagc 1080caggctgctc tgggagacgc aaacgctgac
gccatcggcc gcggaacttt ctacggtaaa 1140acagaggaaa aggagcccag
caagctcgag taa 117322390PRTArtificial SequenceBased on Arabidopsis
thaliana sequence 22Met Val Lys Glu Asp Lys Gln Thr Asp Gly Asp Arg
Trp Arg Gly Leu1 5 10 15Ala Tyr Asp Thr Ser Asp Asp Gln Gln Asp Ile
Thr Arg Gly Lys Gly 20 25 30Met Val Asp Ser Val Phe Gln Ala Pro Met
Gly Thr Gly Thr His His 35 40 45Ala Val Leu Ser Ser Tyr Glu Tyr Val
Ser Gln Gly Leu Arg Gln Tyr 50 55 60Asn Leu Asp Asn Met Met Asp Gly
Phe Tyr Ile Ala Pro Ala Phe Met65 70 75 80Asp Lys Leu Val Val His
Ile Thr Lys Asn Phe Leu Thr Leu Pro Asn 85 90 95Ile Lys Val Pro Leu
Ile Leu Gly Ile Trp Gly Gly Lys Gly Gln Gly 100 105 110Lys Ser Phe
Gln Cys Glu Leu Val Met Ala Lys Met Gly Ile Asn Pro 115 120 125Ile
Met Met Ser Ala Gly Glu Leu Glu Ser Gly Asn Ala Gly Glu Pro 130 135
140Ala Lys Leu Ile Arg Gln Arg Tyr Arg Glu Ala Ala Asp Leu Ile
Lys145 150 155 160Lys Gly Lys Met Cys Cys Leu Phe Ile Asn Asp Leu
Asp Ala Gly Ala 165 170 175Gly Arg Met Gly Gly Thr Thr Gln Tyr Thr
Val Asn Asn Gln Met Val 180 185 190Asn Ala Thr Leu Met Asn Ile Ala
Asp Asn Pro Thr Asn Val Gln Leu 195 200 205Pro Gly Met Tyr Asn Lys
Glu Glu Asn Ala Arg Val Pro Ile Ile Cys 210 215 220Thr Gly Asn Asp
Phe Ser Thr Leu Tyr Ala Pro Leu Ile Arg Asp Gly225 230 235 240Arg
Met Glu Lys Phe Tyr Trp Ala Pro Thr Arg Glu Asp Arg Ile Gly 245 250
255Ile Cys Lys Gly Ile Phe Arg Thr Asp Lys Ile Lys Asp Glu Asp Ile
260 265 270Val Arg Leu Val Asp Gln Phe Pro Gly Gln Ser Ile Asp Phe
Phe Gly 275 280 285Ala Leu Arg Ala Arg Val Tyr Asp Asp Glu Val Arg
Lys Phe Val Glu 290 295 300Ser Leu Gly Val Glu Asn Ile Gly Lys Arg
Leu Val Asn Ser Arg Glu305 310 315 320Gly Pro Pro Val Phe Glu Gln
Pro Glu Met Thr Tyr Glu Lys Leu Met 325 330 335Glu Tyr Gly Asn Met
Leu Val Met Glu Gln Glu Asn Val Lys Arg Val 340 345 350Gln Leu Ala
Glu Thr Tyr Leu Ser Gln Ala Ala Leu Gly Asp Ala Asn 355 360 365Ala
Asp Ala Ile Gly Arg Gly Thr Phe Tyr Gly Lys Thr Glu Glu Lys 370 375
380Glu Pro Ser Lys Leu Glu385 3902317DNAArtificial Sequenceprimer
23cagacaatgt tggcctc 172418DNAArtificial Sequenceprimer
24acgagtaacg atggtagg 182515DNAArtificial Sequenceprimer
25gtctatacct tgagc 152614DNAArtificial Sequenceprimer 26tcagtcatac
tcgg 14
* * * * *