U.S. patent application number 11/961942 was filed with the patent office on 2008-09-04 for materials and methods for the modulation of cyclin-dependent kinase inhibitor-like polypeptides in maize.
Invention is credited to Beth Savidge, Wei Zheng.
Application Number | 20080216193 11/961942 |
Document ID | / |
Family ID | 34083399 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080216193 |
Kind Code |
A1 |
Savidge; Beth ; et
al. |
September 4, 2008 |
MATERIALS AND METHODS FOR THE MODULATION OF CYCLIN-DEPENDENT KINASE
INHIBITOR-LIKE POLYPEPTIDES IN MAIZE
Abstract
Isolated or purified nucleic acid molecules encoding
polypeptides having cyclin-dependent kinase (CDK) inhibitor-like
activity; vectors; host cells; polypeptides having CDK
inhibitor-like activity; nucleic acid constructs for suppression of
CDK inhibitor-like activity; methods of suppressing and
up-regulating the expression of one or more CDK inhibitor-like
genes in a maize cell, tissue, organ, or plant; a maize cell,
tissue, organ, or plant in which the expression of a CDK
inhibitor-like gene has been suppressed or up-regulated in
accordance with such methods; and a seed (and the oil and meal
thereof) obtained from a plant in which the expression of one or
more CDK inhibitor-like genes has been suppressed or
up-regulated.
Inventors: |
Savidge; Beth; (Davis,
CA) ; Zheng; Wei; (Davis, CA) |
Correspondence
Address: |
SONNENSCHEIN NATH & ROSENTHAL LLP
P.O. BOX 061080, SOUTH WACKER DRIVE STATION, SEARS TOWER
CHICAGO
IL
60606
US
|
Family ID: |
34083399 |
Appl. No.: |
11/961942 |
Filed: |
December 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10890629 |
Jul 14, 2004 |
7329799 |
|
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11961942 |
|
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60486935 |
Jul 14, 2003 |
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Current U.S.
Class: |
800/286 ;
435/419; 435/468; 536/23.6; 800/320.1 |
Current CPC
Class: |
C12N 15/8247 20130101;
Y02A 40/146 20180101; C12N 15/8261 20130101; C07K 14/415
20130101 |
Class at
Publication: |
800/286 ;
536/23.6; 435/468; 800/320.1; 435/419 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07H 21/04 20060101 C07H021/04; A01H 5/00 20060101
A01H005/00; C12N 5/04 20060101 C12N005/04 |
Claims
1-7. (canceled)
8. A nucleic acid construct comprising at least one transcribable
nucleotide sequence, the expression of which results in the
suppression of an endogenous nucleic acid selected from the group
consisting of: SEQ ID NO: 1 [ZmKRP1], a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 2 [ZmKRP1], SEQ ID
NO: 3 [ZmKRP2], and a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 4 [ZmKRP2], alone or in further combination
with at least one other nucleotide sequence selected from the group
consisting of SEQ ID NO: 5 [ZmKRP3], a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 6 [ZmKRP3], SEQ ID NO: 7
[ZmKRP4], a nucleotide sequence encoding the amino acid sequence of
SEQ ID NO: 8 [ZmKRP4], SEQ ID NO: 9 [ZmKRP5], a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 10 [ZmKRP5], SEQ ID
NO: 11 [ZmKRP6], a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 12 [ZmKRP6], SEQ ID NO: 13 [ZmKRP7], a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
14 [ZmKRP7], SEQ ID NO: 15 [ZmKRP8], a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 16 [ZmKRP8], SEQ ID NO: 17
[ZmKRP9], and a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 18 [ZmKRP9], wherein, when the nucleic acid
construct comprises two or more transcribable nucleotide sequences,
the nucleotide sequences can be present in any order on the nucleic
acid construct and can be polycistronic, wherein each transcribable
nucleotide sequence comprises at least about 100 contiguous
nucleotides, and wherein the expression of each transcribable
nucleotide sequence optionally induces gene suppression.
9. The nucleic acid construct of claim 8, which further comprises a
promoter that is preferentially expressed in the germ and/or
aleurone of a kernel of maize and the promoter is operably linked
to at least one transcribable nucleotide sequence.
10. The nucleic acid construct of claim 9, wherein the promoter is
an oleosin promoter of maize, a globulin promoter of maize, a MIP
synthase promoter of maize, a Perl promoter of barley, an
embryo-preferred promoter such as P-Os.CPC214 from rice or an
embryo-preferred promoter such as P-Zm.CEP1, P-Zm.CPC214,
P-Zm.CPC214tr1, or P-Zm.CPC214tr2 from maize.
11. The nucleic acid construct of claim 8, wherein the at least one
transcribable nucleotide sequence is a noncoding sequence.
12. The nucleic acid construct of claim 11, wherein the noncoding
sequence is an intron, a nucleotide sequence from a promoter
region, a nucleotide sequence from a 5' untranslated region, a
nucleotide sequence from a 3' untranslated region, or a fragment of
any of the foregoing.
13-22. (canceled)
23. A method of suppressing the expression of one or more CDK
inhibitor-like genes in a maize cell, a maize tissue, a maize
organ, or a maize plant, which method comprises contacting said
maize cell, maize tissue, maize organ or maize plant with a nucleic
acid construct of claims 8, 14, or 18.
24. The method of claim 23, wherein the suppression is accomplished
by antisense suppression, sense suppression, dsRNA, ribozyme, or a
combination thereof.
25. The method of claim 23, wherein the nucleic acid construct
further comprises a promoter that is preferentially expressed in
the germ and/or aleurone of a kernel of maize and the promoter is
operably linked to the at least one nucleotide sequence.
26. The method of claim 25, wherein the promoter is an oleosin
promoter of maize, a globulin promoter of maize, a MIP synthase
promoter of maize, a Perl promoter of barley, an embryo-preferred
promoter such as P-Os.CPC214 from rice or an embryo-preferred
promoter such as P-Zm.CEP1, P-Zm.CPC214, P-Zm.CPC214tr1, or
P-Zm.CPC214tr2 from maize.
27. A maize cell, a maize tissue, a maize organ, or a maize plant
in which the expression of a CDK inhibitor-like gene has been
suppressed in accordance with the method of claim 23.
28. A seed obtained from a plant of claim 27.
29-30. (canceled)
31. The method of claim 23 wherein the suppression results in
increased oil.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/486,935, filed Jul. 14, 2003, which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to isolated or purified
nucleic acid molecules encoding polypeptides having
cyclin-dependent kinase (CDK) inhibitor-like activity,
complementary and antisense nucleic acid molecules, vectors, host
cells, polypeptides having CDK inhibitor-like activity and
materials and methods for suppressing and up-regulating the
expression of one or more CDK inhibitor-like genes in maize.
BACKGROUND OF THE INVENTION
[0003] The activation/inactivation of CDK by cyclins drives the
transition between the different phases of the cell cycle. CDK
activity is also regulated by CDK inhibitors known as KRPs
(Kip-related proteins, wherein "Kip" is short for "kinase inhibitor
proteins"). Other CDK inhibitors from plants and animal systems are
known as CKI, ICK, Cip, and Ink (De Veylder, 2001, Plant Cell,
13:1653-1667) and KIS (Jasinski et al., J. Cell. Sci., 115:973-982
(2002)). CDK inhibitors have been identified in plants, including
Arabidopsis and tobacco. The CDK inhibitors of plants have
approximately 35 amino acids at the carboxy terminus homologous to
the amino-terminal cyclin/CDK-binding domain of animal CDK
inhibitors of the p21.sup.Cip1/p27.sup.Kip1/p57.sup.Kip2 types.
Outside of the carboxy-terminal region, the plant CDK inhibitors
identified thus far are structurally different.
BRIEF SUMMARY OF THE INVENTION
[0004] Suppression of the expression of a CDK inhibitor-like gene
can result in increased cellular proliferation and/or increased
mitotic index. Suppression of the expression of a CDK
inhibitor-like gene can also result in increased oil and increased
yield.
[0005] The present invention provides isolated or purified nucleic
acid molecules encoding CDK inhibitor-like polypeptides from maize.
In one embodiment, the molecule comprises (i) the nucleotide
sequence of SEQ ID NO: 1 [ZmKRP1; "Zm" indicates Zea mays; "KRP"
indicates Kip related protein; the number is the designation given
to a particular gene], (ii) a nucleotide sequence encoding the
amino acid sequence of SEQ ID NO: 2 [ZmKRP1], (iii) a nucleotide
sequence that encodes an amino acid sequence that is at least about
70% identical to the amino acid sequence of SEQ ID NO: 2 [ZmKRP1]
and has CDK inhibitor-like activity, or (iv) a fragment of any of
(i)-(iii), wherein the fragment comprises at least about 35
contiguous nucleotides.
[0006] In another embodiment, the molecule comprises (i) the
nucleotide sequence of SEQ ID NO: 3 [ZmKRP2], (ii) a nucleotide
sequence encoding the amino acid sequence of SEQ ID NO: 4 [ZmKRP2],
(iii) a nucleotide sequence that encodes an amino acid sequence
that is at least about 70% identical to the amino acid sequence of
SEQ ID NO: 4 [ZmKRP2] and has CDK inhibitor-like activity, or (iv)
a fragment of any of (i)-(iii), wherein the fragment comprises at
least about 35 contiguous nucleotides.
[0007] In yet another embodiment, the molecule consists essentially
of (i) the nucleotide sequence of SEQ ID NO: 7 [ZmKRP4], (ii) a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
8 [ZmKRP4], or (iii) a nucleotide sequence that encodes an amino
acid sequence that is at least about 70% identical to the amino
acid sequence of SEQ ID NO: 8 [ZmKRP4] and has CDK inhibitor-like
activity.
[0008] In still yet another embodiment, the molecule comprises (i)
the nucleotide sequence of SEQ ID NO: 9 [ZmKRP5], (ii) a nucleotide
sequence encoding the amino acid sequence of SEQ ID NO: 10
[ZmKRP5], (iii) a nucleotide sequence that encodes an amino acid
sequence that is at least about 70% identical to the amino acid
sequence of SEQ ID NO: 10 [ZmKRP5] and has CDK inhibitor-like
activity, or (iv) a fragment of any of (i)-(iii), wherein the
fragment comprises at least about 120 contiguous nucleotides.
[0009] In a further embodiment, the molecule consists essentially
of (i) the nucleotide sequence of SEQ ID NO: 11 [ZmKRP6], (ii) a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
12 [ZmKRP6], (iii) a nucleotide sequence that encodes an amino acid
sequence that is at least about 70% identical to the amino acid
sequence of SEQ ID NO: 12 [ZmKRP6] and has CDK inhibitor-like
activity, or (iv) a fragment of any of (i)-(iii), wherein the
fragment comprises at least about 390 contiguous nucleotides.
[0010] In view of the above, the present invention further provides
vectors comprising the above-described nucleic acid molecules. Host
cells comprising the isolated or purified nucleic acid molecules,
optionally in the form of vectors, are also provided.
[0011] Also in view of the above, the present invention provides
isolated or purified polypeptides encoded by the above-described
nucleic acid molecules. Isolated or purified nucleic acid
molecules, optionally in the form of a vector, comprising or
consisting essentially of complementary and antisense sequences to
some of the above-described molecules, SEQ ID NOS: 13, 15, and 17,
and nucleotide sequences encoding the amino acid sequences of SEQ
ID NOS: 6, 14, 16, and 18 are also provided. When the sequence is
SEQ ID NO: 1, encodes SEQ ID NO: 2, is SEQ ID NO: 3, or encodes SEQ
ID NO: 4, the complementary or antisense sequence comprises at
least 35 contiguous nucleotides.
[0012] Further provided are nucleic acid constructs comprising at
least one transcribable nucleotide sequence, the expression of
which results in the suppression of an endogenous nucleotide
sequence selected from the group consisting of:
[0013] SEQ ID NO: 1 [ZmKRP1], a nucleotide sequence encoding the
amino acid sequence of SEQ TD NO: 2 [ZmKRP1], SEQ ID NO: 3
[ZmKRP2], and a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 4 [ZmKRP2],
[0014] alone or in further combination with at least one other
nucleotide sequence, the expression of which results in the
suppression of an endogenous nucleotide sequence selected from the
group consisting of SEQ ID NO: 5 [ZmKRP3], a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 6 [ZmKRP3], SEQ ID
NO: 7 [ZmKRP4], a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 8 [ZmKRP4], SEQ ID NO: 9 [ZmKRP5], a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
10 [ZmKRP5], SEQ ID NO: 11 [ZmKRP6], a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 12 [ZmKRP6], SEQ ID NO: 13
[ZmKRP7], a nucleotide sequence encoding the amino acid sequence of
SEQ ID NO: 14 [ZmKRP7], SEQ ID NO: 15 [ZmKRP8], a nucleotide
sequence encoding the amino acid sequence of SEQ ID NO: 16
[ZmKRP8], SEQ ID NO: 17 [ZmKRP9], and a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 18 [ZmKRP9],
[0015] wherein, when the nucleic acid construct comprises two or
more transcribable nucleotide sequences, the nucleotide sequences
can be present in any order on the nucleic acid construct and can
be polycistronic,
[0016] wherein each transcribable nucleotide sequence comprises at
least about 100 contiguous nucleotides, and
[0017] wherein the expression of each transcribable nucleotide
sequence optionally induces gene suppression.
[0018] In another embodiment, the nucleic acid construct comprises
at least one transcribable nucleotide sequence, the expression of
which results in the suppression of an endogenous nucleic acid
sequence selected from SEQ ID NO: 7 [ZmKRP4] and/or SEQ ID NO: 17
[ZmKRP9], either one or both of the transcribable nucleotide
sequence in further combination with at least one other
transcribable nucleotide sequence, the expression of which results
in the suppression of an endogenous nucleotide sequence selected
from the group consisting of: [0019] (i) SEQ ID NO:1 [ZmKRP1] or a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
2 [ZmKRP1], [0020] (ii) SEQ ID NO: 3 [ZmKRP2] or a nucleotide
sequence encoding the amino acid sequence of SEQ ID NO: 4 [ZmKRP2],
[0021] (iii) SEQ ID NO: 5 [ZmKRP3] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 6 [ZmKRP3], provided
that the transcribable nucleotide sequence does not entirely
correspond to nucleotides 175-342 of SEQ ID NO: 5, [0022] (iv) SEQ
ID NO: 9 [ZmKRP5] or a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 10 [ZmKRP5], provided that the transcribable
nucleotide sequence does not entirely correspond to nucleotides
450-570 of SEQ ID NO: 9, [0023] (v) SEQ ID NO: 1 [ZmKRP6] or a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
12 [ZmKRP6], provided that the transcribable nucleotide sequence
does not entirely correspond to nucleotides 380-765 of SEQ ID NO:
11, [0024] (vi) SEQ ID NO: 13 [ZmKRP7] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 14 [ZmKRP7],
provided that the transcribable nucleotide sequence does not
entirely correspond to nucleotides 335-718 of SEQ ID NO: 13, and
[0025] (vii) SEQ ID NO: 15 [ZmKRP8] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 16 [ZmKRP8],
provided that the transcribable nucleotide sequence does not
entirely correspond to nucleotides 350-405 or 420-698 of SEQ ID NO:
15,
[0026] wherein the transcribable nucleotide sequences can be
present in any order on the nucleic acid construct and can be
polycistronic,
[0027] wherein each transcribable nucleotide sequence comprises at
least about 100 contiguous nucleotides, and
[0028] wherein the expression of each transcribable nucleotide
sequence optionally induces gene suppression.
[0029] In yet another embodiment, the nucleic acid construct
comprises at least one transcribable nucleotide sequence, the
expression of which results in the suppression of an endogenous
nucleotide sequence selected from the group consisting of: [0030]
(i) SEQ ID NO: 1 [ZmKRP1] or a nucleotide sequence encoding the
amino acid sequence of SEQ ID NO: 2 [ZmKRP1], [0031] (ii) SEQ ID
NO: 3 [ZmKRP2] or a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 4 [ZmKRP2], [0032] (iii) SEQ ID NO: 5
[ZmKRP3] or a nucleotide sequence encoding the amino acid sequence
of SEQ ID NO: 6 [ZmKRP3], provided that the transcribable
nucleotide sequence does not entirely correspond to nucleotides
175-342 of SEQ ID NO: 5, [0033] (iv) SEQ ID NO: 9 [ZmKRP5] or a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
10 [ZmKRP5], provided that the transcribable nucleotide sequence
does not entirely correspond to nucleotides 450-570 of SEQ ID NO:
9, [0034] (v) SEQ ID NO: 11 [ZmKRP6] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 12 [ZmKRP6],
provided that the transcribable nucleotide sequence of SEQ ID NO:
11 does not entirely correspond to nucleotides 380-765 of SEQ ID
NO: 11, [0035] (vi) SEQ ID NO: 13 [ZmKRP7] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 14 [ZmKRP7],
provided that the transcribable nucleotide sequence does not
entirely correspond to nucleotides 335-718 of SEQ ID NO: 13, [0036]
(vii) SEQ ID NO: 15 [ZmKRP8] or a nucleotide sequence encoding the
amino acid sequence of SEQ ID NO: 16 [ZmKRP8], provided that the
transcribable nucleotide sequence does not entirely correspond to
nucleotides 350-405 or 420-698 of SEQ ID NO: 15, and [0037] (viii)
SEQ ID NO: 17 [ZmKRP9] or a nucleotide sequence encoding the amino
acid sequence of SEQ ID NO: 18 [ZmKRP9], provided that the
transcribable nucleotide sequence does not entirely correspond to
nucleotides 1-183 of SEQ ID NO: 17,
[0038] wherein the transcribable nucleotide sequences can be
present in any order on the nucleic acid construct and can be
polycistronic,
[0039] wherein each transcribable nucleotide sequence comprises at
least about 100 contiguous nucleotides, and
[0040] wherein the expression of each transcribable nucleotide
sequence optionally induces gene suppression.
[0041] A method of suppressing the expression of one or more CDK
inhibitor-like genes in a maize cell, a maize tissue, a maize
organ, or a maize plant is also provided. The method comprises
contacting said maize cell, maize tissue, maize organ or maize
plant with a nucleic acid construct described above.
[0042] In this regard, also provided are a maize cell, a maize
tissue, a maize organ, or a maize plant in which the expression of
a CDK inhibitor-like gene has been suppressed in accordance with
such a method. A seed obtained from a plant in which the expression
of one or more CDK inhibitor-like genes has been suppressed is also
provided.
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 is the amino acid sequence alignment of the
polypeptides of the present invention with published sequences of
CDK inhibitors of Arabidopsis. The sequences of Arabidopsis are as
indicated in Example 1.
[0044] FIG. 2 is the construct map of pMON71270.
[0045] FIG. 3 is the construct map of pMON71279.
[0046] FIG. 4 is a schematic diagram of a dsRNA intermediate
construct showing the primer regions and directions (A) and the
assembling procedures (B).
[0047] FIG. 5 is a schematic diagram of a dsRNA intermediate
construct showing the primer regions and directions (A) and the
assembling procedures (B).
BRIEF DESCRIPTION OF THE SEQUENCES
[0048] SEQ ID NO: 1 is the nucleotide sequence of ZmKRP1. [0049]
SEQ ID NO: 2 is the amino acid sequence of ZmKRP1. [0050] SEQ ID
NO: 3 is the nucleotide sequence of ZmKRP2. [0051] SEQ ID NO: 4 is
the amino acid sequence of ZmKRP2. [0052] SEQ ID NO: 5 is the
nucleotide sequence of ZmKRP3. [0053] SEQ ID NO: 6 is the amino
acid sequence of ZmKRP3. [0054] SEQ ID NO: 7 is the nucleotide
sequence of ZmKRP4. [0055] SEQ ID NO: 8 is the amino acid sequence
of ZmKRP4. [0056] SEQ ID NO: 9 is the nucleotide sequence of
ZmKRP5. [0057] SEQ ID NO: 10 is the amino acid sequence of ZmKRP5.
[0058] SEQ ID NO: 11 is the nucleotide sequence of ZmKRP6. [0059]
SEQ ID NO: 12 is the amino acid sequence of ZmKRP6. [0060] SEQ ID
NO: 13 is the nucleotide sequence of ZmKRP7. [0061] SEQ ID NO: 14
is the amino acid sequence of ZmKRP7. [0062] SEQ ID NO: 15 is the
nucleotide sequence of ZmKRP8. [0063] SEQ ID NO: 16 is the amino
acid sequence of ZmKRP8. [0064] SEQ ID NO: 17 is the nucleotide
sequence of ZmKRP9. [0065] SEQ ID NO: 18 is the amino acid sequence
of ZmKRP9. [0066] SEQ ID NO: 19 is the nucleotide sequence of 200
base pairs (bp) from the 3' end of ZmKRP1 (F1). [0067] SEQ ID NO:
20 is the nucleotide sequence of 200 bp from near the 3' end of
ZmKRP2 (F2). [0068] SEQ ID NO: 21 is the nucleotide sequence of 200
bp from the 3' end of ZmKRP3 (F3). [0069] SEQ ID NO: 22 is the
nucleotide sequence of 200 bp from the 3' end of ZmKRP4 (F4).
[0070] SEQ ID NO: 23 is the nucleotide sequence of 200 bp from the
3' end of ZmKRP5 (F5). [0071] SEQ ID NO: 24 is the nucleotide
sequence of 200 bp from close to the 3' end of ZmKRP6 and ZmKRP7
(F6). [0072] SEQ ID NO: 25 is the nucleotide sequence of 200 bp
from near the 3' end of ZmKRP8 (F7). [0073] SEQ ID NO: 26 is the
nucleotide sequence of 183 bp from the 3' end of ZmKRP8 (F8).
[0074] SEQ ID NO: 27 is the nucleotide sequence of the ZmDnaK
intron fragment. [0075] SEQ ID NO: 28 is the nucleotide sequence of
the primer P1-StuI. [0076] SEQ ID NO: 29 is the nucleotide sequence
of the primer P1-2R. [0077] SEQ ID NO: 30 is the nucleotide
sequence of the primer P2-3. [0078] SEQ ID NO: 31 is the nucleotide
sequence of the primer P4-Bgl. [0079] SEQ ID NO: 32 is the
nucleotide sequence of the primer P3-4. [0080] SEQ ID NO: 33 is the
nucleotide sequence of the primer P5-Bam. [0081] SEQ ID NO: 34 is
the nucleotide sequence of the primer P5-Bgl. [0082] SEQ ID NO: 35
is the nucleotide sequence of the primer P1-SseBam. [0083] SEQ ID
NO: 36 is the nucleotide sequence of the primer Pi-Bgl. [0084] SEQ
ID NO: 37 is the nucleotide sequence of the primer Pi-Bam. [0085]
SEQ ID NO: 38 is the nucleotide sequence of the primer P5-Nco.
[0086] SEQ ID NO: 39 is the nucleotide sequence of the primer P5-6.
[0087] SEQ ID NO: 40 is the nucleotide sequence of the primer P6-7.
[0088] SEQ ID NO: 41 is the nucleotide sequence of the primer
P8-Bgl. [0089] SEQ ID NO: 42 is the nucleotide sequence of the
primer P7-8. [0090] SEQ ID NO: 43 is the nucleotide sequence of the
primer P8-i. [0091] SEQ ID NO: 44 is the nucleotide sequence of the
5' untranslated region (UTR) of ZmKRP1. [0092] SEQ ID NO: 45 is the
nucleotide sequence of the 3' UTR of ZmKRP1. [0093] SEQ ID NO: 46
is the nucleotide sequence of the 5' UTR of ZmKRP2. [0094] SEQ ID
NO: 47 is the nucleotide sequence of the 3' UTR of ZmKRP2. [0095]
SEQ ID NO: 48 is the nucleotide sequence of the 3' UTR of ZmKRP3.
[0096] SEQ ID NO: 49 is the nucleotide sequence of the 5' UTR of
ZmKRP4. [0097] SEQ ID NO: 50 is the nucleotide sequence of the 3'
UTR of ZmKRP4. [0098] SEQ ID NO: 51 is the nucleotide sequence of
the 5' UTR of ZmKRP5. [0099] SEQ ID NO: 52 is the nucleotide
sequence of the 3' UTR of ZmKRP5. [0100] SEQ ID NO: 53 is the
nucleotide sequence of the 5' UTR of ZmKRP6. [0101] SEQ ID NO: 54
is the nucleotide sequence of the 3' UTR of ZmKRP6. [0102] SEQ ID
NO: 55 is the nucleotide sequence of the 5' UTR of ZmKRP7. [0103]
SEQ ID NO: 56 is the nucleotide sequence of the 3' UTR of ZmKRP7.
[0104] SEQ ID NO: 57 is the nucleotide sequence of the 5' UTR of
ZmKRP8. [0105] SEQ ID NO: 58 is the nucleotide sequence of the 3'
UTR of ZmKRP9. [0106] SEQ ID NO: 59 is a conserved sequence from
the C-terminal region of certain polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
[0107] Suppression of the expression of a CDK inhibitor-like gene
can result in increased cellular proliferation and/or increased
mitotic index. For example, increasing cellular proliferation may
result in an increased embryo size and an increase in the amount of
oil.
[0108] Suppression of the expression of a CDK inhibitor-like gene
can also result in increased yield. Many agronomic traits can
affect "yield". For example, these could include, without
limitation, plant height, pod number, pod position on the plant,
number of internodes, incidence of pod shatter, grain size,
efficiency of nodulation and nitrogen fixation, efficiency of
nutrient assimilation, resistance to biotic and abiotic stress,
carbon assimilation, plant architecture, resistance to lodging,
percent seed germination, seedling vigor, and juvenile traits. For
example, these could also include, without limitation, efficiency
of germination (including germination in stressed conditions),
growth rate (including growth rate in stressed conditions), ear
number, seed number per ear, seed size, composition of seed
(starch, oil, protein), characteristics of seed fill. "Yield" can
be measured in may ways, these might include test weight, seed
weight, seed number per plant, seed weight, seed number per unit
area (i.e., seeds, or weight of seeds, per acre), bushels per acre,
tonnes per acre, tons per acre, kilo per hectare. In an embodiment,
a plant of the present invention might exhibit an enhanced trait
that is a component of yield. An enhanced trait is a trait, or
phenotype of a plant, that is changed in a way that could be viewed
as an agronomic improvement when compared to a non-transgenic plant
of the same, or very similar, genotype.
[0109] The present invention provides, among other things, isolated
or purified nucleic acid molecules encoding polypeptides of maize
having CDK inhibitor-like activity. By "CDK-inhibitor like
activity" is meant the ability to reduce CDK activity in a histone
H1 kinase assay (see, Example 14). Preferably, CDK activity is
reduced by at least about 10%, more preferably at least about 20%,
even more preferably at least about 30%, still even more preferably
at least about 40%, 50%, 60%, 70%, or 80%, and most preferably at
least about 90% or more. By "isolated" is meant having been removed
from its natural environment. If a nucleic acid, isolated means the
separation of a nucleic acid from other nucleic acid molecules and
the substantial separation from other cellular material and either
culture medium, if produced by recombinant techniques, or chemical
precursors, if synthesized. By "purified" is meant having been
increased in purity, wherein "purity" is a relative term, and is
not to be construed as absolute purity. "Nucleic acid molecules" is
intended to encompass a polymer of DNA (e.g., cDNA or genomic DNA)
or RNA (e.g., mRNA), i.e., a polynucleotide, which can be
single-stranded or double-stranded, which can comprise chimeric
DNA/RNA oligonucleotides, and which can contain non-natural or
altered nucleotides.
[0110] In one embodiment, an isolated or purified nucleic acid
molecule comprising (i) the nucleotide sequence of SEQ ID NO: 1
[ZmKRP1], (ii) a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 2 [ZmKRP1], (iii) a nucleotide sequence that
encodes an amino acid sequence that is at least about 70% (or 75%,
80%, 85%, 90%, 95%, or 99%) identical to the amino acid sequence of
SEQ ID NO: 2 [ZmKRP1] and has CDK inhibitor-like activity, or (iv)
a fragment of any of (i)-(iii), wherein the fragment comprises at
least about 35 (or 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, or more) contiguous nucleotides, is provided.
[0111] In another embodiment, an isolated or purified nucleic acid
molecule comprising (i) the nucleotide sequence of SEQ ID NO: 3
[ZmKRP2], (ii) a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 4 [ZmKRP2], (iii) a nucleotide sequence that
encodes an amino acid sequence that is at least about 70% (or 75%,
80%, 85%, 90%, 95%, or 99%) identical to the amino acid sequence of
SEQ ID NO: 4 [ZmKRP2] and has CDK inhibitor-like activity, or (iv)
a fragment of any of (i)-(iii), wherein the fragment comprises at
least about 35 (or 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, or more) contiguous nucleotides, is provided.
[0112] In yet another embodiment, the present invention provides an
isolated or purified nucleic acid molecule consisting essentially
of (i) the nucleotide sequence of SEQ ID NO: 7 [ZmKRP4], (ii) a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
8 [ZmKRP4], or (iii) a nucleotide sequence that encodes an amino
acid sequence that is at least about 70% (or 75%, 80%, 85%, 90%,
95%, or 99%) identical to the amino acid sequence of SEQ ID NO: 8
[ZmKRP4] and has CDK inhibitor-like activity.
[0113] In still yet another embodiment, the present invention
provides an isolated or purified nucleic acid molecule comprising
(i) the nucleotide sequence of SEQ ID NO: 9 [ZmKRP5], (ii) a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
10 [ZmKRP5], (iii) a nucleotide sequence that encodes an amino acid
sequence that is at least about 70% (or 75%, 80%, 85%, 90%, 95%, or
99%) identical to the amino acid sequence of SEQ ID NO: 10 [ZmKRP5]
and has CDK inhibitor-like activity, or (iv) a fragment of any of
(i)-(iii), wherein the fragment comprises at least about 120 (or
125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,
190, 195, 200, or more) contiguous nucleotides.
[0114] An isolated or purified nucleic acid molecule comprising (i)
the nucleotide sequence of SEQ ID NO: 11 [ZmKRP6], (ii) a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
12 [ZmKRP6], (iii) a nucleotide sequence that encodes an amino acid
sequence that is at least about 70% (or 75%, 80%, 85%, 90%, 95%, or
99%) identical to the amino acid sequence of SEQ ID NO: 12 [ZmKRP6]
and has CDK inhibitor-like activity, and (iv) a fragment of any of
(i)-(iv), wherein the fragment comprises at least about 390 (or
400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460,
465, 470, 475, 480, 485, 490, 495, 500, or more) contiguous
nucleotides, are also provided.
[0115] Such nucleic acid molecules can be amplified using cDNA,
mRNA or genomic DNA as a template and appropriate oligonucleotide
primers according to standard PCR amplification techniques.
Alternatively, they can be synthesized using standard synthetic
techniques, such as an automated DNA synthesizer.
[0116] The above isolated or purified nucleic acid molecules are
characterized, in part, in terms of "percentage of sequence
identity." In this regard, a given nucleic acid molecule can be
compared to an above-described nucleic acid molecule (i.e., the
reference sequence) by optimally aligning the nucleic acid
sequences over a comparison window, wherein the portion of the
polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence, which does not comprise additions or deletions, for
optimal alignment of the two sequences. The percentage of sequence
identity is calculated by determining the number of positions at
which the identical nucleic acid base occurs in both sequences,
i.e., the number of matched positions, dividing the number of
matched positions by the total number of positions in the window of
comparison, and multiplying the result by 100 to yield the
percentage of sequence identity. Optimal alignment of sequences for
comparison can be conducted by computerized implementations of
known algorithms. Sequence alignments and percent identity
calculations can be performed by the Clustal Algorithm (Higgins et
al., CABIOS, 5 (2): 151-153 (1989)), using default parameters of
the Megalign program of the LASARGENE bioinformatics computing
suite (DNASTAR Inc., Madison, Wis.), hereinafter referred to as
"Clustal W Algorithm." Other computerized implementations include
GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Package
(Accelrys, San Diego, Calif.), or BlastN and BlastX available from
the National Center for Biotechnology Information, Bethesda, Md.).
In addition, alignment can be performed by inspection. Sequences
are typically compared using BESTFIT or BlastN with default
parameters. A preferred method of determining identity is Clustal W
Algorithm, in accordance with the parameters set forth in Example
1.
[0117] One of ordinary skill in the art will appreciate, however,
that two polynucleotide sequences can be substantially different at
the nucleic acid level, yet encode substantially similar, if not
identical, amino acid sequences, due to the degeneracy of the
genetic code. The present invention is intended to encompass such
polynucleotide sequences. In this regard, it should be noted that
fragments of the above-described nucleic acid molecules can be used
as probes to identify other polynucleotide sequences.
[0118] An isolated or purified nucleic acid molecule comprising or
consisting essentially of the complementary or antisense sequence
(or fragment thereof, such as one comprising at least about 35
contiguous nucleotides) to an above-described isolated or purified
nucleic acid molecule, as well as to SEQ ID NOS: 13, 15, and 17,
and nucleotide sequences encoding the amino acid sequences of SEQ
ID NOS: 6, 14, 16, and 18 are also provided. The isolated or
purified nucleic acid molecule is optionally in the form of a
vector.
[0119] If desired, an above-described isolated or purified nucleic
acid molecule can be operably linked to a promoter. Promoters
include, but are not limited to, promoters that function in
bacteria, bacteriophage, plastids or plant cells, including
promoters that are preferentially expressed in the male
reproductive tissue. Any suitable promoter can be used as herein
described below. Examples of such promoters include, but are not
limited to, the SILKY1 promoter of maize (Ambrose et al., Molec.
Cell, 5(3):569-579 (2000)), the NTM19 promoter of tobacco (Oldenhof
et al., Plant Molec. Biol., 31:213-225 (1996); U.S. Pat. No.
6,407,314; and WO 97/30166), the NPG1 promoter of tobacco (see,
e.g., U.S. Application Publication 2003/0061635), the PCA55
promoter of maize (see, e.g., WO 92/13957), the AP3 promoter of
Arabidopsis (Zhou et al., Planta, 215:248-257 (2002)), and the Bgp1
promoter of Brassica (Zhou et al. (2002), infra). Other examples
include an anther-preferred promoter, such as that from LAT52
(Twell et al., Molec. Gen. Genet., 217(2-3):240-245 (1989); and
Twell et al., Genes Dev., 5(3):496-507 (1991)) or RA8 of rice (Jeon
et al., Plant Mol. Biol., 39(1):35-44 (1999)), a pollen-preferred
promoter, such as that from maize Zm13 (Guerrero et al., Molec.
Gen. Genet., 224:161-168 (1993)) or rice PSI (Zou et al., Amer. J.
Bot., 81(5):552-561 (1994)), and a microspore-preferred promoter,
such as that from apg (anther-preferred; Twell et al., Sex. Plant.
Repro., 6:217-224 (1993)). In some instances, a promoter that is
preferentially expressed in the male reproductive tissue of maize
can be desirable (see, e.g., Example 5).
[0120] If desired, an above-described isolated or purified nucleic
acid molecule can be operably linked to a promoter. Promoters of
the instant invention generally include, but are not limited to,
promoters that function in bacteria, bacteriophage, or plant cells.
Useful promoters for bacterial expression are the lacZ, Sp6, T7,
T5, or E. coli glg C promoters. Useful promoters for plants cells
include the globulin promoter (see, for example, Belanger and Kriz,
Genet., 129:863-872 (1991)), gamma zein Z27 promoter (see, for
example, Lopes et al., Mol. Gen. Genet., 247:603-613 (1995)), L3
oleosin promoter (U.S. Pat. No. 6,433,252), barley PER1 promoter
(Stacey et al., Plant Mol. Biol., 31:1205-1216 (1996), embryo
preferred promoters such as P-Zm.CEP1, P-Zm.CPC214, P-Zm.CPC214tr1,
P-Zm.CPC214tr2 or P-Os.CPC214 (U.S. Provisional Application No.
60/531,483), incorporated herein by reference, USP promoters (U.S.
Application Publication No. 2003/229918), incorporated herein by
reference, 7S.alpha. promoter, 7S.alpha.' promoter (see, e.g.,
Beachy et al., EMBO J., 4:3047 (1985) or Schuler et al., Nucleic
Acid Res., 10(24):8225-8244 (1982)), CaMV 35S promoter (Odell et
al., Nature, 313:810 (1985)), the CaMV 19S (Lawton et al., Plant
Mol. Biol., 9:31F (1987)), nos (Ebert et al., PNAS U.S.A., 84:5745
(1987)), Adh (Walker et al., PNAS U.S.A., 84:6624 (1987)), sucrose
synthase (Yang et al., PNAS U.S.A. 87:4144 (1990)), tubulin, actin
(Wang et al., Mol. Cell. Biol., 12:3399 (1992)), cab (Sullivan et
al., Mol. Gen. Genet., 215:431 (1989)), PEPCase promoter (Hudspeth
et al., Plant Mol. Biol., 12:579 (1989)), or those associated with
the R gene complex (Chandler et al., The Plant Cell, 1: 1175
(1989)). The Figwort Mosaic-Virus (FMV) promoter (Richins et al.,
Nucleic Acids Res., 20:8451 (1987)), arcelin, tomato E8, patatin,
ubiquitin, mannopine synthase (mas), soybean seed protein glycinin
(Gly), and soybean vegetative storage protein (vsp) promoters are
other examples of useful promoters.
[0121] In one preferred embodiment the promoter used is highly
expressed in the germ and/or aleurone tissue. Preferred promoters
known to function in maize, and in other plants, include the
promoters for oleosins (for example, the L3 promoter, U.S. Pat. No.
6,433,252), the globulin promoter (see, for example, Belanger and
Kriz, Genet., 129:863-872, 1991), the barley peroxiredoxin promoter
(Perl, Stacy et al., Plant Mol. Biol., 31:1205-1216, Accession
#X96551), embryo preferred promoters such as P-ZM.CEP1,
P-Zm.CPC214, P-Zm.CPC214tr1, P-Zm.CPC214tr2, or P-Os.CPC214 (U.S.
Provisional Application No. 60/531,483), incorporated herein by
reference, or the MIP synthase promoter of maize (WO 01/40440
A2).
[0122] Examples of promoters highly expressed in the endosperm
include promoters from genes encoding zeins, which are a group of
storage proteins found in maize endosperm. Genomic clones for zein
genes have been isolated (Pedersen et al., Cell, 29: 1015-1026
(1982) and Russell et al., Transgenic Res., 6(2): 157-168 (1997))
and the promoters from these clones, including the 15 kD, 16 kD, 19
kD, 22 kD, and 27 kD genes, can be used. Other preferred promoters,
known to function in maize, and in other plants, include the
promoters for the following genes: waxy (granule bound starch
synthase), Brittle and Shrunken 2 (ADP glucose pyrophosphorylase),
Shrunken l (sucrose synthase), branching enzymes I and II, starch
synthases, debranching enzymes, oleosins, glutelins, sucrose
synthases (Yang et al., PNAS U.S.A., 87:4144-4148 (1990)), BetlI
(basal endosperm transfer layer) and globulin 1. Other promoters
useful in the practice of the invention that are known by one of
skill in the art are also contemplated by the invention.
[0123] Moreover, transcription enhancers or duplications of
enhancers can be used to increase expression from a particular
promoter. Examples of such enhancers include, but are not limited
to the Adh intron1 (Callis et al., Genes Develop., 1:1183 (1987)),
a rice actin intron (McElroy et al., Mol. Gen. Genet.,
231(1):150-160 (1991)) (U.S. Pat. No. 5,641,876), sucrose synthase
intron (Vasil et al., Plant Physiol., 91:5175 (1989)), a maize
HSP70 intron (Rochester et al., EMBO J., 5:451-458 (1986)) a TMV
omega element (Gallie et al., The Plant Cell, 1:301 (1999)) the
CaMV 35S enhancer or an octopine synthase enhancer (Last et al.,
U.S. Pat. No. 5,290,924). As the DNA sequence between the
transcription initiation site and the start of the coding sequence,
i.e., the untranslated leader sequence, can influence gene
expression, one may also wish to employ a particular leader
sequence. Any leader sequence available to one of skill in the art
may be employed. Preferred leader sequences direct optimum levels
of expression of the attached gene, for example, by increasing or
maintaining mRNA stability and/or by preventing inappropriate
initiation of translation (Joshi, Nucl. Acid Res., 15:6643 (1987)).
The choice of such sequences is at the discretion of those of skill
in the art. Sequences that are derived from genes that are highly
expressed in higher plants, and in soybean, corn, rice, and canola
in particular, are contemplated.
[0124] An inducible promoter can be turned on or off by an
exogenously added agent so that expression of an operably linked
nucleic acid is also turned on or off. For example, a bacterial
promoter, such as the P.sub.tac, promoter can be induced to varying
levels of gene expression depending on the level of
isothiopropylgalactoside added to the transformed bacterial cells.
In plants, inducible promoters can be used in those instances where
the expression of a given gene is desired after a host plant has
reached maturity. Such inducible promoters include heat shock
promoters, stress response promoters, and chemically inducible
promoters.
[0125] Expression cassettes of the invention will also include a
sequence near the 3' end of the cassette that acts as a signal to
terminate transcription from a heterologous nucleic acid and that
directs polyadenylation of the resultant mRNA. These are commonly
referred to as 3' untranslated regions or 3' UTRs. Some 3' elements
that can act as transcription termination signals include those
from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan
et al., Nucl. Acid Res., 11:369 (1983)), a napin 3' untranslated
region (Kridl et al., Seed Sci Res., 1:209-219 (1991)), a globulin
3' untranslated region (Belanger and Kriz, Genetics, 129:863-872
(1991)), or one from a zein gene, such as Z27 (Lopes et al., Mol
Gen Genet., 247:603-613 (1995)). Other 3' elements known by one of
skill in the art also can be used in the vectors of the
invention.
[0126] The present invention further provides a vector comprising
an above-described isolated or purified nucleic acid molecule. A
nucleic acid molecule as described above can be cloned into any
suitable vector and can be used to transform or transfect any
suitable host. The selection of vectors and methods to construct
them are commonly known to persons of ordinary skill in the art and
are described in general technical references (see, in general,
"Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and
Grossman, eds., Academic Press (1987)). Desirably, the vector
comprises regulatory sequences, such as transcription and
translation initiation and termination codons, which are specific
to the type of host (e.g., bacterium, fungus, or plant) into which
the vector is to be introduced, as appropriate and taking into
consideration whether the vector is DNA or RNA.
[0127] Constructs of vectors, which are circular or linear, can be
prepared to contain an entire nucleic acid sequence as described
above or a portion thereof ligated to a replication system
functional in a prokaryotic or eukaryotic host cell. Replication
systems can be derived from ColE1, 2 m.mu. plasmid, .lamda. phage,
f1 filamentous phage, Agrobacterium species (e.g., Ag. tumefaciens
and Ag. rhizogenes), and the like.
[0128] In addition to the replication system and the inserted
nucleic acid, the construct can include one or more marker genes,
which allow for selection of transformed or transfected hosts.
Marker genes include biocide resistance, e.g., resistance to
antibiotics, heavy metals, herbicides; etc., complementation in an
auxotrophic host to provide prototrophy, and the like.
[0129] Suitable vectors include those designed for propagation or
expression or both. A preferred cloning vector is selected from the
group consisting of the pUC series, the pBluescript series
(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison,
Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and
the pEX series (Clonetech, Palo Alto, Calif.). Bacteriophage
vectors, such as .lamda.GT10, .lamda.GT11, .lamda.ZapII
(Stratagene), .lamda. EMBL4, and .lamda. NM1149, also can be used.
Examples of plant expression vectors include pBI101, pBI101.2,
pBI101.3, pBI121, and pBIN19 (Clonetech, Palo Alto, Calif.).
Examples of yeast expression vectors include pYES2.1, pYES2, and
other pYES derivatives (Invitrogen, Carlsbad, Calif.).
[0130] A plant expression vector can comprise a native or normative
promoter operably linked to an above-described nucleic acid
molecule. The selection of promoters, e.g., strong, weak,
inducible, tissue-specific (i.e., specifically or preferentially
expressed in a tissue), organ-specific (i.e., specifically or
preferentially expressed in an organ) and developmental-specific
(i.e., specifically or preferentially expressed during a particular
stage(s) of development), is within the skill in the art.
Similarly, the combining of a nucleic acid molecule as described
above with a promoter is also within the skill in the art (see,
e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989)).
[0131] If it is desired to up-regulate the expression of a CDK
inhibitor-like gene, i.e., to increase the expression of a CDK
inhibitor-like gene by any means, such as by about two-fold,
ten-fold, 100-fold, or more at the mRNA level or protein level, it
is preferred to do so by introducing a gene encoding a CDK
inhibitor-like polypeptide as provided herein. The gene is
preferably introduced by way of a vector. It is preferred that a
lot of inhibitor is expressed in the tissue of interest. Multiple
extra copies of the gene can be introduced into the plant cell,
plant tissue, plant organ, or plant or a vector comprising a strong
promoter, such as promoter, which is preferentially or specifically
expressed in male reproductive tissue, is introduced into the plant
cell, plant tissue, plant organ, or plant such that the gene is
expressed at a higher rate, thereby generating more mRNA, which, in
turn, is translated into more of the encoded protein.
[0132] As used herein "suppression" or "suppressing" means any of
the well-known methods for suppressing a transcript or a protein
from a gene including post-transcriptional gene suppression and
transcriptional suppression. Post-transcriptional gene suppression
is mediated by transcribed RNA having homology to a gene targeted
for suppression. The RNA transcribed from the suppressing transgene
preferably has a double stranded component to effect what is called
RNA interference (RNAi). Transcriptional suppression is mediated by
a transcribed double-stranded RNA having homology to promoter DNA
sequence to effect what is called promoter trans-suppression.
[0133] More particularly, post-transcriptional gene suppression by
double-stranded RNA can result from plant transformation with
anti-sense DNA constructs as disclosed by Shewmaker et al. (U.S.
Pat. Nos. 5,107,065 and 5,759,829), from plant transformation with
a sense-oriented DNA construct as disclosed by Jorgensen et al.
(U.S. Pat. Nos. 5,283,184 and 5,231,020), or with an RNAi construct
as disclosed by Redenbaugh et al. (Safety Assessment of Genetically
Engineered Flavr Savr.TM. Tomato, CRC Press, Inc., 1992), by
Goldbach et al. (EP 0426195 A1, 1991), by Sijen et al. (The Plant
Cell, 8:2277-2294, 1996), by Waterhouse et al.
(WO 99/53050), by Graham et al. (WO 99/49029), by Lowe et al. (U.S.
Application Publication No. 2003/0175965 A1), by Fillatti (U.S.
patent application Ser. No. 10/465,800), by Plaetinck et al. (U.S.
Application Publication No. 2003/0061626 A1), by Liu et al. (U.S.
Pat. No. 6,326,193), by Agrawal et al. (WO 94/01550), by Werner et
al. (WO 98/05770), by Oeller (U.S. Application Publication No.
2002/0048814 A1), by Gutterson et al. (U.S. Application Publication
No. 2003/0018993 A1), and by Glassman et al. (U.S. Application
Publication No. 2003/0036197 A1). All of the above-described
patents, applications, and international publications disclosing
materials and methods for post-transcriptional gene suppression in
plants are incorporated herein by reference.
[0134] Transcriptional suppression such as promoter
trans-suppression can be effected by expressing a DNA construct
comprising a promoter operably linked to inverted repeats of
promoter DNA for a target gene. Constructs useful for such gene
suppression mediated by promoter trans-suppression are disclosed by
Mette et al., The EMBO Journal, 18(1):241-148, 1999 and Mette et
al., The EMBO Journal, 19(19):5194-5201, 2000, both of which are
incorporated herein by reference.
[0135] A preferred method of post-transcriptional gene suppression
in plants employs either sense-oriented or antisense-oriented,
transcribed RNA which is stabilized, e.g., with a terminal hairpin
structure. A preferred DNA construct for effecting
post-transcriptional gene suppression is transcribed to a segment
of antisense oriented RNA having homology to a gene targeted for
suppression, where the antisense RNA segment is followed at the 3'
end by a contiguous, complementary, shorter segment of RNA in the
sense orientation. The use of self-stabilized antisense RNA
oligonucleotides in plants is disclosed in WO 94/01550 (Agrawal et
al.). See also WO 98/05770 (Werner et al.) where the antisense RNA
is stabilized by hairpin forming repeats of poly(CG) nucleotides.
See also U.S. Application Publication No. 2002/0048814 A1 (Oeller)
where sense or antisense RNA is stabilized by a poly(T)-poly(A)
tail. See also U.S. Application Publication No. 2003/0018993 A1
(Gutterson et al.) where sense or antisense RNA is stabilized by an
inverted repeat of a subsequence of an NOS gene. See also U.S.
Application Publication No. 2003/0036197 A1 (Glassman et al.) where
RNA having homology to a target is stabilized by 2 complementary
RNA regions. All of the above-described patents, applications, and
international publications disclosing materials and methods for
employing stabilized RNA and its use in gene suppression in plants
are incorporated herein by reference.
[0136] Transcriptional suppression such as promoter
trans-suppression can be effected by expressing a DNA construct
comprising a promoter operably linked to inverted repeats of
promoter DNA for a target gene. Constructs useful for such gene
suppression mediated by promoter trans-suppression are disclosed by
Mette et al., The EMBO Journal, 18(1):241-148, 1999 and Mette et
al., The EMBO Journal, 19(19):5194-5201, 2000, both of which are
incorporated herein by reference.
[0137] Still yet another method is the use of a dominant negative
mutant. For example, a dominant negative mutant of a polypeptide
having CDK inhibitor-like activity as described herein can be
generated by completely or partially deleting the C-terminal coding
sequence, in particular all or part of the C-terminal coding
sequence that is highly conserved among the polypeptides described
herein. The resulting mutant can be operably linked to a promoter,
such as an embryo-specific promoter from maize, for example, and
cloned into a vector for introduction into a corn plant or part
thereof. See, e.g., Jasinski et al., Plant Physiol., 130:1871-1882
(2002)).
[0138] Ribozymes also have been reported to have use as a means to
inhibit expression of endogenous plant genes (see, e.g., Merlo et
al., Plant Cell, 10(10): 1603-1622 (1998)). It is possible to
design ribozymes that specifically pair with virtually any target
RNA and cleave the phosphodiester backbone at a specific location,
thereby functionally inactivating the target RNA. In carrying out
this cleavage, the ribozyme is not itself altered and is, thus,
capable of recycling and cleaving other molecules, making it a true
enzyme. The inclusion of ribozyme sequences within antisense RNAs
confers RNA-cleaving activity upon them, thereby increasing the
activity of the constructs. The design and use of target
RNA-specific ribozymes is described in Haseloff et al., Nature,
334:585-591 (1988). Preferably, the ribozyme comprises at least
about 20 continuous nucleotides complementary to the target
sequence on each side of the active site of the ribozyme.
[0139] Alternatively, reverse genetics systems, which are
well-known in the art, can be used to generate and isolate
suppressed or null mutants. One such system, the Trait Utility
System for Corn, i.e., TUSC, is based on successful systems from
other organisms (Ballinger et al., PNAS U.S.A., 86:9402-9406
(1989); Kaiser et al., PNAS U.S.A. 87:1686-1690 (1990); and
Rushforth et al., Mol. Cell. Biol., 13:902-910 (1993)). The central
feature of the system is to identify Mu transposon insertions
within a DNA sequence of interest in anticipation that at least
some of these insertion alleles will be mutants. To develop the
system in corn, DNA was collected from a large population of
Mutator transposon stocks that were then self-pollinated to produce
F2 seed. To find Mu transposon insertions within a specified DNA
sequence, the collection of DNA samples is screened via PCR using a
gene-specific primer and a primer that anneals to the inverted
repeats of Mu transposons. A PCR product is expected only when the
template DNA comes from a plant that contains a Mu transposon
insertion within the target gene. Once such a DNA sample is
identified, F2 seed from the corresponding plant is screened for a
transposon insertion allele. Transposon insertion mutations of the
an1 gene have been obtained via the TUSC procedure (Bensen et al.,
Plant Cell, 7:75-84 (1995)). This system is applicable to other
plant species, at times modified as necessary in accordance with
knowledge and skill in the art.
[0140] T-DNA insertional mutagenesis can be used to generate
insertional mutations in one of the above-mentioned genes so as to
affect adversely the expression of a given gene. T-DNA tagged lines
of plants can be screened using PCR. For example, a primer can be
designed for one end of the T-DNA and another primer can be
designed for the gene of interest and both primers can be used in
PCR. If no PCR product is obtained, then there is no insertion in
the gene of interest. In contrast, if a PCR product is obtained,
then there is an insertion in the gene of interest. Insertional
mutations, however, often generate null alleles, which can be
lethal. Alternatively, if there is more than one gene that encodes
for a given enzyme, a mutation in one of the genes may not result
in decreased expression of the enzyme encoded by the gene.
[0141] Another alternative method to decrease expression of a given
gene is to use a compound that inhibits expression of one of the
above-mentioned genes or that inhibits the activity of the protein
encoded by one of the above-mentioned genes. In this regard, x-ray
or gamma radiation can be used as can chemical mutagens, such as
ethyl methyl sulfonate (EMS) or dimethyl butyric acid (DMB).
[0142] In addition to the above, gene replacement technology can be
used to increase or decrease expression of a given gene. Gene
replacement technology is based upon homologous recombination (see,
Schnable et al., Curr. Opinions Plant Biol., 1: 123 (199.8)). The
nucleic acid of the enzyme of interest can be manipulated by
mutagenesis (e.g., insertions, deletions, duplications, or
replacements) to either increase or decrease enzymatic function.
The altered sequence can be introduced into the genome to replace
the existing, e.g., wild-type, gene via homologous recombination
(Puchta and Hohn, Trends Plant Sci., 1:340 (1996); Kempin et al.,
Nature, 389:802 (1997)).
[0143] The activity of a given CDK inhibitor-like polypeptide can
be measured in vitro. For example, the ability of a given CDK
inhibitor-like polypeptide to prevent phosphorylation of H1
histones by CDK can be measured (see, e.g., Example 14).
[0144] Whether or not a given CDK inhibitor-like polypeptide
affects the expression of other genes can be determined using a
transgenic plant, for example. As microarray chip technology
becomes established and available (DeRisi et al., Science,
278:680-686 (1997)), the effect of a given genetic alteration on
the expression of all identified genes of maize can be determined
using microarray chips. Metabolic radiotracer studies can be
performed to measure the generation of different product pools in
vivo. In such studies, radioactively labeled precursors are
provided to intact tissues and the radioactive label is monitored
as the precursor is metabolized. By comparing wild-type plants and
plants that have reduced activities of one of the CDK
inhibitor-like polypeptide genes, the effect of the reduction in a
given CDK inhibitor-like polypeptide can be determined.
[0145] In view of the above, the present invention provides a host
cell comprising an above-described isolated or purified nucleic
acid molecule, optionally in the form of a vector. Suitable hosts
include bacteria, yeast and plant cells, including E. coli, B.
subtilis, A. tumefaciens, S. cerevisiae, and N. crassa. E. coli
hosts include TB-1, TG-2, DH5.alpha., XL-Blue MRF' (Stratagene,
Austin, Tex.), SA2821, Y1090, and TG02. Plant cells include maize
cells.
[0146] Also in view of the above, the present invention provides an
isolated or purified polypeptide encoded by an above-described
isolated or purified nucleic acid molecule. The polypeptide
preferably comprises an amino end and a carboxyl end. The
polypeptide can comprise D-amino acids, L-amino acids, or a mixture
of D- and L-amino acids. The D-form of the amino acids, however, is
particularly preferred, since a protein comprised of D-amino acids
is expected to have a greater retention of its biological activity
in vivo, given that the D-amino acids are not recognized by
naturally occurring proteases.
[0147] Alterations of the native amino acid sequence to produce
variant polypeptides can be done by a variety of means known to
those ordinarily skilled in the art. For instance, amino acid
substitutions can be conveniently introduced into the polypeptides
at the time of synthesis. Alternatively, site-specific mutations
can be introduced by ligating into an expression vector a
synthesized oligonucleotide comprising the modified site.
Alternately, oligonucleotide-directed, site-specific mutagenesis
procedures can be used, such as disclosed in Walder et al., Gene,
42:133 (1986); Bauer et al., Gene, 37:73 (1985); and U.S. Pat. Nos.
4,518,584 and 4,737,462.
[0148] It is within the skill of the ordinary artisan to select
synthetic and naturally-occurring amino acids that effect
conservative or neutral substitutions for any particular
naturally-occurring amino acids. The ordinarily skilled artisan
desirably will consider the context in which any particular amino
acid substitution is made, in addition to considering the
hydrophobicity or polarity of the side-chain, the general size of
the side chain and the pK value of side-chains with acidic or basic
character under physiological conditions. For example, lysine,
arginine, and histidine are often suitably substituted for each
other, and more often arginine and histidine. As is known in the
art, this is because all three amino acids have basic side chains,
whereas the pK value for the side-chains of lysine and arginine are
much closer to each other (about 10 and 12) than to histidine
(about 6). Similarly, glycine, alanine, valine, leucine, and
isoleucine are often suitably substituted for each other, with the
proviso that glycine is frequently not suitably substituted for the
other members of the group. This is because each of these amino
acids is relatively hydrophobic when incorporated into a
polypeptide, but glycine's lack of an .alpha.-carbon allows the phi
and psi angles of rotation (around the .alpha.-carbon) so much
conformational freedom that glycinyl residues can trigger changes
in conformation or secondary structure that do not often occur when
the other amino acids are substituted for each other. Other groups
of amino acids frequently suitably substituted for each other
include, but are not limited to, the group consisting of glutamic
and aspartic acids; the group consisting of phenylalanine,
tyrosine, and tryptophan; and the group consisting of serine,
threonine, and optionally, tyrosine. Additionally, the ordinarily
skilled artisan can readily group synthetic amino acids with
naturally-occurring amino acids.
[0149] If desired, the polypeptides can be modified, for instance,
by glycosylation, amidation, carboxylation, or phosphorylation, or
by the creation of acid addition salts, amides, esters, in
particular C-terminal esters, and N-acyl derivatives of the
polypeptides of the invention. The polypeptides also can be
modified to create protein derivatives by forming covalent or
noncovalent complexes with other moieties in accordance with
methods known in the art. Covalently-bound complexes can be
prepared by linking the chemical moieties to functional groups on
the side chains of amino acids comprising the polypeptides, or at
the N- or C-terminus. Desirably, such modifications and
conjugations do not adversely affect the activity of the
polypeptides (and variants thereof). While such modifications and
conjugations can have greater or lesser activity, the activity
desirably is not negated and is characteristic of the unaltered
polypeptide.
[0150] The polypeptides (and fragments, variants, and fusion
proteins) can be prepared by any of a number of conventional
techniques. The polypeptide can be isolated or purified from a
naturally occurring source or from a recombinant source. For
instance, in the case of recombinant proteins, a DNA fragment
encoding a desired protein can be subcloned into an appropriate
vector using well-known molecular genetic techniques (see, e.g.,
Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed.
(Cold Spring Harbor Laboratory, 1989) and other references cited
herein under "EXAMPLES"). The fragment can be transcribed and the
protein subsequently translated in vitro. Commercially available
kits also can be employed (e.g., such as manufactured by Clontech,
Palo Alto, Calif.; Amersham Life Sciences, Inc., Arlington Heights,
Ill.; InVitrogen, San Diego, Calif., and the like). The polymerase
chain reaction optionally can be employed in the manipulation of
nucleic acids.
[0151] Such polypeptides also can be synthesized using an automated
peptide synthesizer in accordance with methods known in the art.
Alternately, the polypeptide (and fragments, variants, and fusion
proteins) can be synthesized using standard peptide synthesizing
techniques well-known to those of ordinary skill in the art (e.g.,
as summarized in Bodanszky, Principles of Peptide Synthesis,
(Springer-Verlag, Heidelberg: 1984)). In particular, the
polypeptide can be synthesized using the procedure of solid-phase
synthesis (see, e.g., Merrifield, J. Am. Chem. Soc., 85:2149-54
(1963); Barany et al., Int. J. Peptide Protein Res., 30:705-739
(1987); and U.S. Pat. No. 5,424,398). If desired, this can be done
using an automated peptide synthesizer. Removal of the
t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc)
amino acid blocking groups and separation of the protein from the
resin can be accomplished by, for example, acid treatment at
reduced temperature. The polypeptide-containing mixture then can be
extracted, for instance, with diethyl ether, to remove non-peptidic
organic compounds, and the synthesized protein can be extracted
from the resin powder (e.g., with about 25% w/v acetic acid).
Following the synthesis of the polypeptide, further purification
(e.g., using HPLC) optionally can be done in order to eliminate any
incomplete proteins, polypeptides, peptides, or free amino acids.
Amino acid and/or HPLC analysis can be performed on the synthesized
polypeptide to validate its identity. For other applications
according to the invention, it may be preferable to produce the
polypeptide as part of a larger fusion protein, either by chemical
conjugation, or through genetic means, such as are known to those
ordinarily skilled in the art. In this regard, the present
invention also provides a fusion protein comprising the isolated or
purified polypeptide (or fragment thereof) or variant thereof and
one or more other polypeptides/protein(s) having any desired
properties or effector functions.
[0152] The nucleic acid molecules, vectors, and polypeptides can be
used in agricultural methods and various screening assays (see,
e.g., WO 02/28893 and U.S. Pat. No. 6,215,048). For example, a
nucleic acid molecule can be used to express, e.g., via a vector, a
CDK inhibitor-like polypeptide in a host cell, to detect mRNA
encoding a CDK inhibitor-like polypeptide in a biological sample,
to detect a genetic alteration in a gene encoding a CDK
inhibitor-like polypeptide, such as via a Southern blot, to
suppress a CDK inhibitor-like polypeptide, or to up-regulate a CDK
inhibitor-like polypeptide so as to render a plant male-sterile or
a dwarf, or to inhibit the formation of certain organs, for
example. The polypeptides can be used to compensate for
deficiencies in CDK inhibitor-like polypeptides or for the presence
of mutated CDK inhibitor-like polypeptides having reduced or no
activity in a maize plant, or to treat excessive levels of
substrates, whether direct or indirect, for CDK inhibitor-like
polypeptides in a maize plant. Alternatively, the polypeptides can
be used to screen agents for the ability to modulate their
activity.
[0153] In view of the above, the present invention provides various
nucleic acid constructs, which can be used to suppress a CDK
inhibitor-like polypeptide. In one embodiment, the nucleic acid
construct comprises at least one transcribable nucleotide sequence,
the expression of which results in the suppression of an endogenous
nucleotide sequence selected from the group consisting of:
[0154] SEQ ID NO: 1 [ZmKRP1], a nucleotide sequence encoding the
amino acid sequence of SEQ ID NO: 2 [ZmKRP1], SEQ ID NO: 3
[ZmKRP2], and a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 4 [ZmKRP2],
[0155] alone or in further combination with at least one other
transcribable nucleotide sequence, the expression of which results
in the suppression of an endogenous nucleotide sequence selected
from the group consisting of SEQ ID NO: 5 [ZmKRP3], a nucleotide
sequence encoding the amino acid sequence of SEQ ID NO: 6 [ZmKRP3],
SEQ ID NO: 7 [ZmKRP4], a nucleotide sequence encoding the amino
acid sequence of SEQ ID NO: 8 [ZmKRP4], SEQ ID NO: 9 [ZmKRP5], a
nucleotide sequence encoding: the amino acid sequence of SEQ ID NO:
10 [ZmKRP5], SEQ ID NO: 11 [ZmKRP6], a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 12 [ZmKRP6], SEQ ID NO: 13
[ZmKRP7], a nucleotide sequence encoding the amino acid sequence of
SEQ ID NO: 14 [ZmKRP7], SEQ ID NO: 15 [ZmKRP8], a nucleotide
sequence encoding the amino acid sequence of SEQ ID NO: 16
[ZmKRP8], SEQ ID NO: 17 [ZmKRP9], and a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 18 [ZmKRP9],
[0156] wherein, when the nucleic acid construct comprises two or
more transcribable nucleotide sequences, the nucleotide sequences
can be present in any order on the nucleic acid construct and can
be polycistronic, wherein each transcribable nucleotide sequence
comprises at least about 100 (or 125, 150, 175, 200, 225, 250, 275,
300, 325, 375, 400, 425, 450, 500, 750, 1,000, 1,500, 2,000, or
more) contiguous nucleotides, and wherein the expression of each
transcribable nucleotide sequence optionally induces gene
suppression. By "optionally" is meant that each transcribable
nucleotide sequence, independent of any and all other nucleotide
sequences present on the construct, either induces gene suppression
or does not induce gene suppression.
[0157] Alternatively, the nucleic acid construct comprises
transcribable nucleotide sequences, the expression of which results
in the suppression of endogenous nucleotide sequences:
[0158] (i) SEQ ID NO: 1 [ZmKRP1] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 2 [ZmKRP1],
[0159] (ii) SEQ ID NO: 3 [ZmKRP2] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 4 [ZmKRP2],
[0160] (iii) SEQ ID NO: 5 [ZmKRP3] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 6 [ZmKRP3],
[0161] (iv) SEQ ID NO: 7 [ZmKRP4] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 8 [ZmKRP4],
[0162] (v) SEQ ID NO: 9 [ZmKRP5] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 10 [ZmKRP5],
[0163] (vi) SEQ ID NO: 11 [ZmKRP6] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 12 [ZmKRP6],
[0164] (vii) SEQ ID NO: 13 [ZmKRP7] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 14 [ZmKRP7].
[0165] (viii) SEQ ID NO:15 [ZmKRP8] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 16 [ZmKRP8], and
[0166] (ix) SEQ ID NO: 17 [ZmKRP9] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 18 [ZmKRP9].
[0167] In another embodiment, the nucleic acid construct comprises
at least one transcribable nucleotide sequence, the expression of
which results in the suppression of an endogenous nucleotide
sequence of SEQ ID NO: 7 [ZmKRP4] and/or SEQ ID NO: 17 [ZmKRP9],
either one or both in further combination with at least one other
transcribable nucleotide sequence, the expression of which results
in the suppression of an endogenous nucleotide sequence selected
from the group consisting of:
[0168] (i) SEQ ID NO: 1 [ZmKRP1] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 2 [ZmKRP2],
[0169] (ii) SEQ ID NO: 3 [ZmKRP2] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 4 [ZmKRP2],
[0170] (iii) SEQ ID NO: 5 [ZmKRP3] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 6 [ZmKRP3], provided
that the transcribable nucleotide sequence does not entirely
correspond to nucleotides 175-342 of SEQ ID NO: 5,
[0171] (iv) SEQ ID NO: 9 [ZmKRP5] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 10 [ZmKRP5], provided that
the transcribable nucleotide sequence does not entirely correspond
to nucleotides 450-570 of SEQ ID NO: 9,
[0172] (v) SEQ ID NO: 11 [ZmKRP6] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 12 [ZmKRP6], provided that
the transcribable nucleotide sequence does not entirely correspond
to nucleotides 380-765 of SEQ ID NO: 11,
[0173] (vi) SEQ ID NO: 13 [ZmKRP7] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 14 [ZmKRP7],
provided that the transcribable nucleotide sequence does not
entirely correspond to nucleotides 335-718 of SEQ ID NO: 13,
and
[0174] (vii) SEQ ID NO: 15 [ZmKRP8] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 16 [ZmKRP8],
provided that the transcribable nucleotide sequence does not
entirely correspond to nucleotides 350-405 or 420-698 of SEQ ID NO:
15,
[0175] wherein the transcribable nucleotide sequences can be
present in any order on the nucleic acid construct and can be
polycistronic, wherein each transcribable nucleotide sequence
comprises at least about 100 (or 125, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 475, 500, 750, 1,000, 1,500,
2,000, or more) contiguous nucleotides, and wherein the expression
of each transcribable nucleotide sequence optionally induces gene
suppression. As used herein, gene suppression refers to the
reduction of gene expression through various techniques, including,
but not limited to, sense suppression, antisense, dsRNA, and
ribozyme technologies.
[0176] In yet another embodiment, the nucleic acid construct
comprises at least one transcribable nucleotide sequence, the
expression of which results in the suppression of an endogenous
nucleotide sequence selected from the group consisting of:
[0177] (i) SEQ ID NO: 1 [ZmKRP1] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 2 [ZmKRP1],
[0178] (ii) SEQ ID NO: 3 [ZmKRP2] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 4 [ZmKRP2],
[0179] (iii) SEQ ID NO: 5 [ZmKRP3] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 6 [ZmKRP3], provided
that the transcribable nucleotide sequence does not entirely
correspond to nucleotides 175-342 of SEQ ID NO: 5,
[0180] (iv) SEQ ID NO: 9 [ZmKRP5] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 10 [ZmKRP5], provided that
the transcribable nucleotide sequence does not entirely correspond
to nucleotides 450-570 of SEQ ID NO: 9,
[0181] (v) SEQ ID NO: 11 [ZmKRP6] or a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 12 [ZmKRP6], provided that
the transcribable nucleotide sequence of SEQ ID NO: 11 does not
entirely correspond to nucleotides 380-765 of SEQ ID NO: 11,
[0182] (vi) SEQ ID NO: 13 [ZmKRP7] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 14 [ZmKRP7],
provided that the transcribable nucleotide sequence does not
entirely correspond to nucleotides 335-718 of SEQ ID NO: 13,
[0183] (vii) SEQ ID NO: 15 [ZmKRP8] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 16 [ZmKRP8],
provided that the transcribable nucleotide sequence does not
entirely correspond to nucleotides 350-405 or 420-698 of SEQ ID NO:
15, and
[0184] (viii) SEQ ID NO: 17 [ZmKRP9] or a nucleotide sequence
encoding the amino acid sequence of SEQ ID NO: 18 [ZmKRP9],
provided that the transcribable nucleotide sequence does not
entirely correspond to nucleotides 1-183 of SEQ ID NO: 17,
[0185] wherein the transcribable nucleotide sequences can be
present in any order on the nucleic acid construct and can be
polycistronic, wherein each transcribable nucleotide sequence
comprises at least about 100 (or 125, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 500, 750, 1,000, 1,500, 2,000,
or more) contiguous nucleotides, and wherein the expression of each
transcribable nucleotide sequence optionally induces gene
silencing. By "does not entirely correspond" means that at least
one, preferably at least about three nucleotides correspond to
nucleotides outside of the designated ranges of nucleotides.
With respect to the above nucleic acid constructs, that at least
one transcribable nucleotide sequence, the expression of which
results in suppression, can be from a noncoding sequence. For
example, the noncoding sequence can be an intron, a nucleotide
sequence from a promoter region, a nucleotide sequence from a 5'
untranslated region, a nucleotide sequence from a 3' untranslated
region, or a fragment of any of the foregoing. Examples of such
sequences include those of SEQ ID NOS: 44-58.
[0186] The above constructs are exemplary and are not intended to
be limiting. In the suppression constructs, for example, ZmKRP1 or
ZmKRP2 can be inhibited alone or together. In this regard, ZmKRP1
or ZmKRP2 can be inhibited alone or together, in further
combination with any one of ZmKRP3, ZmKRP4, ZmKRP5, ZmKRP6, ZmKRP7,
ZmKRP8, and ZmKRP9. For the sake of convenience, the abbreviation
"ZmKRP" has been deleted from the following lists of combinations.
For example, 1, 2 and 3; 1, 2 and 4; 1, 2 and 5; 1, 2 and 6; 1, 2
and 7; 1, 2 and 8; 1, 2 and 9; 1 and 3; 1 and 4; 1 and 5; 1 and 6;
1 and 7; 1 and 8; 1 and 9; 2 and 3; 2 and 4; 2 and 5; 2 and 6; 2
and 7; 2 and 8; 2 and 9; 1, 3 and 4; 1, 3 and 5; 1, 3 and 6; 1, 3
and 7; 1, 3 and 8; 1, 3 and 9; 2, 3 and 4; 2, 3 and 5; 2, 3 and 6;
2, 3 and 7; 2, 3 and 8; 2, 3 and 9; 1, 4 and 5; 1, 4 and 6; 1, 4
and 7; 1, 4 and 8; 1, 4 and 9; 1, 5 and 6; 1, 5 and 7; 1, 5 and 8;
1, 5 and 9; 2, 4 and 5; 2, 4 and 6; 2, 4 and 7; 2, 4 and 8; 2, 4
and 9; 1, 5 and 6; 1, 5 and 7; 1, 5 and 8; 1, 5 and 9; 2, 5 and 6;
2, 5 and 7; 2, 5 and 8; 2, 5 and 9; 1, 6 and 7; 1, 6 and 8; 1, 6
and 9; 2, 6 and 7; 2, 6 and 8; 2, 6 and 9; 1, 7 and 8; 1, 7 and 9;
2, 7 and 8; 2, 7 and 9; 1, 8 and 9; 2, 8 and 9; 1, 3, 4, and 5; 1,
3, 4, and 6; 1, 3, 4, and 7; 1, 3, 4, and 8; 1, 3, 4, and 9; 2, 3,
4, and 5; 2, 3, 4, and 6; 2, 3, 4, and 7; 2, 3, 4, and 8; 2, 3, 4,
and 9; 1, 4, 5, and 6; 1, 4, 5, and 7; 1, 4, 5, and 8; 1, 4, 5, and
9; 2, 4, 5, and 7; 2, 4, 5, and 8; 2, 4, 5, and 9; 1, 5, 6, and 7;
1, 5, 6, and 8; 1, 5, 6, and 9; 2, 5, 6, and 7; 2, 5, 6, and 8; 2,
5, 6, and 9; 1, 6, 7, and 8; 1, 6, 7, and 9; 2, 6, 7, and 8; 2, 6,
7, and 9; 1, 7, 8, and 9; 2, 7, 8, and 9; 1, 3, 4, 5, and 6; 1, 3,
4, 5, and 7; 1, 3, 4, 5, and 8; 1, 3, 4, 5, and 9; 2, 3, 4, 5, and
6; 2, 3, 4, 5 and 7; 2, 3, 4, 5, and 8; 2, 3, 4, 5, and 9; 1, 3, 4,
5, 6, and 7; 1, 3, 4, 5, 6, and 8; 1, 3, 4, 5, 6, and 9; 2, 3, 4,
5, 6, and 7; 2, 3, 4, 5, 6, and 8; 2, 3, 4, 5, 6, and 9; 1, 3, 4,
5, 6, 7, and 8; 1, 3, 4, 5, 6, 7, and 9; 2, 3, 4, 5, 6, 7, and 8;
2, 3, 4, 5, 6, 7, and 9; 1, 3, 4, 5, 6, 7, 8, and 9; 2, 3, 4, 5, 6,
7, 8, and 9; 1, 2, 3, and 4; 1, 2, 3, and 5; 1, 2, 3, and 6; 1, 2,
3, and 7; 1, 2, 3, and 8; 1, 2, 3, and 9; 1, 2, 4, and 5; 1, 2, 4,
and 6; 1, 2, 4, and 7; 1, 2, 4, and 8; 1, 2, 4, and 9; 1, 2, 5, and
6; 1, 2, 5, and 7; 1, 2, 5, and 8; 1, 2, 5, and 9; 1, 2, 6, and 7;
1, 2, 6, and 8; 1, 2, 6, and 9; 1, 2, 7, and 8; 1, 2, 7, and 9; 1,
2, 8, and 9; 1, 2, 3, 4, and 5; 1, 2, 3, 4, and 6; 1, 2, 3, 4, and
7; 1, 2, 3, 4, and 8; 1, 2, 3, 4, and 9; 1, 2, 4, 5, and 6; 1, 2,
4, 5, and 7; 1, 2, 4, 5, and 8; 1, 2, 4, 5, and 9; 1, 2, 5, 6, and
7; 1, 2, 5, 6, and 8; 1, 2, 5, 6, and 9; 1, 2, 6, 7, and 8; 1, 2,
6, 7, and 9; 1, 2, 7, 8, and 9; 1, 2, 3, 4, 5, and 6; 1, 2, 3, 4,
5, and 7; 1, 2, 3, 4, 5, and 8; 1, 2, 3, 4, 5, and 9; 1, 2, 3, 4,
5, 6, and 7; 1, 2, 3, 4, 5, 6, and 8; 1, 2, 3, 4, 5, 6, and 9; 1,
2, 3, 4, 5, 6, 7, and 8; 1, 2, 3, 4, 5, 6, 7, and 9; or 1, 2, 3, 4,
5, 6, 7, 8, and 9 can be inhibited. Furthermore, the constructs can
comprise the nucleotide sequences in any order. The nucleotide
sequences can also vary in length.
[0187] In the suppression constructs, ZmKRP4 or ZmKRP9 can be
inhibited alone or together. In this regard, ZmKRP4 or ZmKRP9 can
be inhibited alone or together, in further combination with any one
of ZmKRP1, ZmKRP2, ZmKRP3, ZmKRP5, ZmKRP6, ZmKRP7, and ZmKRP8. For
example, 4 and 1; 4 and 2; 4 and 3; 4 and 5; 4 and 6; 4 and 7; 4
and 8; 4, 1 and 2; 4, 1 and 3; 4, 1 and 5; 4, 1 and 6; 4, 1 and 7;
4, 1 and 8; 4, 1, 2 and 3; 4, 1, 2, and 5; 4, 1, 2, and 6; 4, 1, 2,
and 7; 4, 1, 2, and 8; 4, 1, 2, 3, and 5; 4, 1, 2, 3, and 6; 4, 1,
2, 3, and 7; 4, 1, 2, 3, and 8; 4, 1, 2, 3, 5, and 6; 4, 1, 2, 3,
5, and 7; 4, 1, 2, 3, 5, and 8; 4, 1, 2, 3, 5, 6, and 7; 4, 1, 2,
3, 5, 6, and 8; 4, 1, 2, 3, 5, 6, 7, and 8; 4, 2 and 3; 4, 2 and 5;
4, 2 and 6; 4, 2 and 7; 4, 2 and 8; 4, 3 and 5; 4, 3 and 6; 4, 3
and 7; 4, 3 and 8; 4, 5 and 6; 4, 5 and 7; 4, 5 and 8; 4, 6 and 7;
4, 6 and 8; 4, 7 and 8; 4, 2, 3, and 5; 4, 2, 3, and 6; 4, 2, 3,
and 7; 4, 2, 3, and 8; 4, 3, 5, and 6; 4, 3, 5, and 7; 4, 3, 5, and
8; 4, 3, 6, and 7; 4, 3, 6, and 8; 4, 5, 6, and 7; 4, 5, 6, and 8;
4, 6, 7, and 8; 4, 2, 3, 5, and 6; 4, 2, 3, 5, and 7; 4, 2, 3, 5,
and 8; 4, 2, 3, 5, 6, and 7; 4, 2, 3, 5, 6, and 8; 4, 2, 3, 5, 6,
7, and 8; 9 and 1; 9 and 2; 9 and 3; 9 and 5; 9 and 6; 9 and 7; 9
and 8; 9, 1 and 2; 9, 1 and 3; 9, 1 and 5; 9, 1 and 6; 9, 1 and 7;
9, 1 and 8; 9, 1, 2, and 3; 9, 1, 2, and 5; 9, 1, 2, and 6; 9, 1,
2, and 7; 9, 1, 2, and 8; 9, 1, 2, 3, and 5; 9, 1, 2, 3, and 6; 9,
1, 2, 3, and 7; 9, 1, 2, 3, and 8; 9, 1, 2, 3, 5, and 6; 9, 1, 2,
3, 5, and 7; 9, 1, 2, 3, 5, and 8; 9, 1, 2, 3, 5, 6, and 7; 9, 1,
2, 3, 5, 6, and 8; 9, 1, 2, 3, 5, 6, 7, and 8; 9, 2 and 3; 9, 2 and
5; 9, 2 and 6; 9, 2 and 7; 9, 2 and 8; 9, 3 and 5; 9, 3 and 6; 9, 3
and 7; 9, 3 and 8; 9, 5 and 6; 9, 5, and 7; 9, 5, and 8; 9, 6 and
7; 9, 6 and 8; 9, 7 and 8; 9, 2, 3, and 5; 9, 2, 3, and 6; 9, 2, 3,
and 7, 9, 2, 3, and 8; 9, 3, 5, and 6; 9, 3, 5, and 7; 9, 3, 5, and
8; 9, 3, 6, and 7; 9, 3, 6, and 8; 9, 5, 6, and 7; 9, 5, 6 and 8;
9, 6, 7, and 8; 9, 2, 3, 5, and 6; 9, 2, 3, 5, and 7; 9, 2, 3, 5,
and 8; 9, 2, 3, 5, 6, and 7; 9, 2, 3, 5, 6, and 8; 9, 2, 3, 5, 6,
7, and 8; 4, 9 and 1; 4, 9 and 2; 4, 9 and 3; 4, 9 and 5; 4, 9 and
6; 4, 9 and 7; 4, 9 and 8; 4, 9, 1, and 2; 4, 9, 1, and 3; 4, 9, 1,
and 5; 4, 9, 1, and 6; 4, 9, 1, and 7; 4, 9, 1, and 8; 4, 9, 1, 2,
and 3; 4, 9, 1, 2, and 5; 4, 9, 1, 2, and 6; 4, 9, 1, 2 and 7; 4,
9, 1, 2, and 8; 4, 9, 1, 2, 3, and 5; 4, 9, 1, 2, 3, and 6; 4, 9,
1, 2, 3, and 7; 4, 9, 1, 2, 3, and 8; 4, 9, 1, 2, 3, 5, and 6; 4,
9, 1, 2, 3, 5, and 7; 4, 9, 1, 2, 3, 5, and 8; 4, 9, 1, 2, 3, 5, 6,
and 7; 4, 9, 1, 2, 3, 5, 6, and 8; 4, 9, 1, 2, 3, 5, 6, 7, and 8;
4, 9, 2, and 3; 4, 9, 2, 4, 9, 3; and 8; 4, 9, 5, and 6; 4, 9, 5,
and 7; 4, 9, 5, and 8; 4, 9, 6, and 7; 4, 9, 5, 6, and 8; 4, 9, 7,
and 8; 4, 9, 2, 3, and 5; 4, 9, 2, 3, and 6; 4, 9, 2, 3, 5, and 7;
4, 9, 2, 3, and 8; 4, 9, 3, 5, and 6; 4, 9, 3, 5, and 7; 4, 9, 3,
5, and 8; 4, 9, 3, 6, and 7; 4, 9, 3, 6, and 8; 4, 9, 5, 6, and 7;
4, 9, 5, 6, 9, 3, 5, and 7; 4, 9, 3, 6, 7, and 8; 4, 9, 2, 3, 6,
and 6; 4, 9, 2, 3, 6, and 7; 4, 9, 2, 3, 6, and 7; 4, 9, 2, 3, 5,
6, and 7; 4, 9, 2, 3, 5, 6, and 8; or 4, 9, 2, 3, 5, 6, 7, and 8
can be inhibited. Furthermore, the constructs can comprise the
nucleotide sequences in any order. The nucleotide sequences can
also vary in length.
[0188] Any of the above constructs can further comprise a promoter,
which is preferentially or specifically expressed in the germ
and/or aleurone of a kernel of maize and the promoter is operably
linked to at least one nucleotide sequence. The promoter can be an
oleosin promoter, such as L3, a globulin promoter, or a Perl
promoter, for example. In the above suppression constructs, the at
least one transcribable nucleotide sequence, the expression of
which results in suppression, may be a noncoding sequence. The
noncoding sequence can be an intron, a nucleotide sequence from a
promoter region, a nucleotide sequence from a 5' untranslated
region, a nucleotide sequence from a 3' untranslated region, or a
fragment of any of the foregoing. Examples of such sequences
include those of SEQ ID NOS: 44-58.
[0189] The above constructs can be made in accordance with methods
known in the art (see, in general, "Recombinant DNA Part D,"
Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic
Press (1987) and the references cited herein under "EXAMPLES").
Suitable methods of construction are exemplified in the Examples
set forth herein.
[0190] Techniques for contacting a plant cell, a plant tissue, a
plant organ or a plant with a nucleic acid construct, such as a
vector, so that the nucleic acid construct is taken up by a plant
cell, alone or as part of a plant tissue, a plant organ, or a
plant, and expressed therein are known in the art. Such methods
involve plant tissue culture techniques, for example. Herein,
"contacting" is intended to mean that the cell, tissue, organ, or
plant is brought into contact with the nucleic acid construct or
vector in such a manner that the vector enters the cell and is
expressed therein.
[0191] The plant cell, plant tissue, plant organ, or plant can be
contacted with the vector by any suitable means as known in the
art. Preferably, a transgenic plant expressing the desired protein
is to be produced. Various methods for the introduction of a
desired polynucleotide sequence encoding the desired protein into
plant cells include, but are not limited to: (1) physical methods
such as microinjection (Capecchi, Cell, 22(2):479-488 (1980)),
electroporation (Fromm et al., PNAS U.S.A., 82(17):5824-5828
(1985); U.S. Pat. No. 5,384,253) and microprojectile mediated
delivery (biolistics or gene gun technology) (Christou et al.,
Bio/Technology, 9:957 (1991); Fynan et al., PNAS U.S.A.
90(24):11478-11482 (1993)); (2) virus mediated delivery methods
(Clapp, Clin. Perinatol., 20(1):155-168 (1993); Lu et al., J. Exp.
Med., 178(6):2089-2096 (1993); Eglitis and Anderson, Biotechniques,
6(7):608-614 (1988); and (3) Agrobacterium-mediated transformation
methods; (Fraley et al., PNAS U.S.A., 80:4803 (1983).
[0192] The most commonly used methods for transformation of plant
cells are the Agrobacterium-mediated DNA transfer process) and the
biolistics or microprojectile bombardment mediated process (i.e.,
the gene gun) Agrobacterium-mediated transformation is achieved
through the use of a genetically engineered soil bacterium
belonging to the genus Agrobacterium. Several Agrobacterium species
mediate the transfer of a specific DNA known as "T-DNA," which can
be genetically engineered to carry any desired piece of DNA into
many plant species. The major events marking the process of T-DNA
mediated pathogenesis are: induction of virulence genes, processing
and transfer of T-DNA. This process is the subject of many reviews
(Ream, Ann. Rev. Phytopathol., 27:583-618 (1989); Howard and
Citovsky, Bioassays, 12:103-108 (1990); Kado, Crit. Rev. Plant
Sci., 10:1-32 (1991); Zambryski, Annual Rev. Plant Physiol. Plant
Mol. Biol., 43:465-490 (1992); Gelvin, In Transgenic Plants, Kung
and Wu, eds., Academic Press, San Diego, pp. 49-87 (1993); Binns
and Howitz, In Bacterial Pathogenesis of Plants and Animals (Dang,
ed.). Berlin: Springer Verlag, pp. 119-138 (1994); Hooykaas and
Beijersbergen, Ann. Rev. Phytopathol., 32:157-179 (1994); Lessl and
Lanka, Cell, 77:321-324 (1994); and Zupan and Zambryski, Annual
Rev. Phytopathol., 27:583-618 (1995)).
[0193] Agrobacterium-mediated genetic transformation of plants
involves several steps. The first step, in which the virulent
Agrobacterium and plant cells are first brought into contact with
each other, is generally called "inoculation." The
Agrobacterium-containing solution is then removed from contact with
the explant by draining or aspiration. Following the inoculation,
the Agrobacterium and plant cells/tissues are permitted to be grown
together for a period of several hours to several days or more
under conditions suitable for growth and
T-DNA transfer. This step is termed "co-culture." Following
co-culture and T-DNA delivery, the plant cells are treated with
bactericidal or bacteriostatic agents to kill the Agrobacterium
remaining in contact with the explant and/or in the vessel
containing the explant. If this is done in the absence of any
selective agents to promote preferential growth of transgenic
versus non-transgenic plant cells, then this is typically referred
to as the "delay" step. If done in the presence of selective
pressure favoring transgenic plant cells, then it is referred to as
a "selection" step. When a "delay" is used, it is typically
followed by one or more "selection" steps. Both the "delay" and
"selection" steps typically include bactericidal or bacteriostatic
agents to kill any remaining Agrobacterium cells because the growth
of Agrobacterium cells is undesirable after the infection
(inoculation and co-culture) process.
[0194] A number of wild-type and disarmed strains of Agrobacterium
tumefaciens and Agrobacterium rhizogenes harboring Ti or Ri
plasmids can be used for gene transfer into plants. The
Agrobacterium hosts contain disarmed Ti and Ri plasmids that do not
contain the oncogenes that cause tumorigenesis or rhizogenesis,
respectively, which are used as the vectors and contain the genes
of interest that are subsequently introduced into plants. Preferred
strains include, but are not limited to, Agrobacterium tumefaciens
strain C58, a nopaline-type strain that is used to mediate the
transfer of DNA into a plant cell, octopine-type strains such as
LBA4404 or succinamopine-type strains, e.g., EHA101 or EHA105. The
nucleic acid molecule, prepared as a DNA composition in vitro, is
introduced into a suitable host such as E. coli and mated into the
Agrobacterium, or directly transformed into competent
Agrobacterium. These techniques are well-known to those of skill in
the art.
[0195] The Agrobacterium can be prepared either by inoculating a
liquid such as Luria Burtani (LB) media directly from a glycerol
stock or streaking the Agrobacterium onto a solidified media from a
glycerol stock, allowing the bacteria to grow under the appropriate
selective conditions, generally from about 26.degree. C.-30.degree.
C., or about 28.degree. C., and taking a single colony or a small
loop of Agrobacterium from the plate and inoculating a liquid
culture medium containing the selective agents. Those of skill in
the art are familiar with procedures for growth and suitable
culture conditions for Agrobacterium as well as subsequent
inoculation procedures. The density of the Agrobacterium culture
used for inoculation and the ratio of Agrobacterium cells to
explant can vary from one system to the next, and, therefore,
optimization of these parameters for any transformation method is
expected.
[0196] Typically, an Agrobacterium culture is inoculated from a
streaked plate or glycerol stock and is grown overnight and the
bacterial cells are washed and resuspended in a culture medium
suitable for inoculation of the explant.
[0197] With respect to microprojectile bombardment (U.S. Pat. Nos.
5,550,318; 5,538,880; 5,610,042; and WO 95/06128; each of which is
specifically incorporated herein by reference in its entirety),
particles are coated with nucleic acids and delivered into cells by
a propelling force. Exemplary particles include those comprised of
tungsten, platinum, and preferably, gold. It is contemplated that,
in some instances, DNA precipitation onto metal particles would not
be necessary for DNA delivery to a recipient cell using
microprojectile bombardment. However, it is contemplated that
particles can contain DNA, rather than be coated with DNA. Hence,
it is proposed that DNA-coated particles can increase the level of
DNA delivery via particle bombardment but are not, in and of
themselves, necessary.
[0198] For the bombardment, cells in suspension are concentrated on
filters or solid culture medium. Alternatively, immature embryos or
other target cells may be arranged on solid culture medium. The
cells to be bombarded are positioned at an appropriate distance
below the macroprojectile stopping plate.
[0199] An illustrative embodiment of a method for delivering DNA
into plant cells by acceleration is the Biolistics Particle
Delivery System (BioRad, Hercules, Calif.), which can be used to
propel particles coated with DNA or cells through a screen, such as
a stainless steel or Nytex screen, onto a filter surface covered
with monocot plant cells cultured in suspension. The screen
disperses the particles so that they are not delivered to the
recipient cells in large aggregates. It is believed that a screen
intervening between the projectile apparatus and the cells to be
bombarded reduces the size of projectiles aggregate and may
contribute to a higher frequency of transformation by reducing the
damage inflicted on the recipient cells by projectiles that are too
large.
[0200] Microprojectile bombardment techniques are widely
applicable, and can be used to transform virtually any plant
species. Examples of species that have been transformed by
microprojectile bombardment include monocot species, such as maize
(WO 95/06128), barley, wheat (U.S. Pat. No. 5,563,055, specifically
incorporated herein by reference in its entirety), rice, oat, rye,
sugarcane, and sorghum; as well as a number of dicots including
tobacco, soybean (U.S. Pat. No. 5,322,783, specifically
incorporated herein by reference in its entirety), sunflower,
peanut, cotton, tomato, and legumes in general (U.S. Pat. No.
5,563,055, specifically incorporated herein by reference in its
entirety).
[0201] To select or score for transformed plant cells regardless of
transformation methodology, the DNA introduced into the cell
contains a gene that functions in a regenerable plant tissue to
produce a compound that confers upon the plant tissue resistance to
an otherwise toxic compound. Genes of interest for use as a
selectable, screenable, or scorable marker include, but are not
limited to, GUS, green fluorescent protein (GFP), luciferase (LUX),
antibiotic or herbicide tolerance genes. Examples of antibiotic
resistance genes include the penicillins, kanamycin (and neomycin,
G418, and bleomycin); methotrexate (and trimethoprim);
chloramphenicol; kanamycin and tetracycline.
[0202] Particularly preferred selectable marker genes for use in
the present invention include genes that confer resistance to
compounds such as antibiotics, like kanamycin (nptII), hygromycin B
(aph IV), and gentamycin (aac3 and aacC4) (Dekeyser et al., Plant
Physiol., 90:217-223 (1989)), and herbicides, like glyphosate
(Della-Cioppa et al., Bio/Technology, 5:579-584 (1987)). Other
selection devices also can be implemented including, but not
limited to, tolerance to phosphinothricin, bialaphos, and
positive-selection mechanisms (Joersbo et al., Mol. Breed.,
4:111-117 (1998)) and are considered within the scope of the
present invention.
[0203] Transformed plant cells, which are derived by any of the
above transformation techniques, can be cultured to regenerate a
whole plant, which possesses the desired transformed phenotype. Any
suitable plant culture medium can be used. Examples of suitable
media would include but are not limited to MS-based media
(Murashige and Skoog, Physiol. Plant, 15:473-497, 1962) or N6-based
media (Chu et al., Scientia Sinica, 18:659, 1975) supplemented with
additional plant growth regulators including but not limited to
auxins, cytokinins, ABA, and gibberellins. Those of skill in the
art are familiar with the variety of tissue culture media, which
when supplemented appropriately, support plant tissue growth and
development and are suitable for plant transformation and
regeneration. These tissue culture media can either be purchased as
a commercial preparation, or custom prepared and modified. Those of
skill in the art are aware that media and media supplements such as
nutrients and growth regulators for use in transformation and
regeneration and other culture conditions such as light intensity
during incubation, pH, and incubation temperatures that can be
optimized for the particular variety of interest.
[0204] One of ordinary skill will appreciate that, after an
expression cassette 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.
[0205] In view of the foregoing, the present invention provides a
method of suppressing the expression of one or more CDK
inhibitor-like genes in a maize cell, a maize tissue, a maize
organ, or a maize plant. The method comprises contacting said maize
cell, maize tissue, maize organ, or maize plant with a nucleic acid
construct as described above.
[0206] Thus, the present invention further provides a maize cell, a
maize tissue, a maize organ, or a maize plant in which the
expression of a CDK inhibitor-like gene has been suppressed in
accordance with such a method. Also provided is a seed obtained
from such a plant and the oil and meal obtained from such a
seed.
[0207] The present invention also provides a container of over
about 10,000, more preferably about 20,000, and even more
preferably about 40,000 seeds where over about 10%, more preferably
about 25%, more preferably about 50%, and even more preferably
about 75% or more preferably about 90% of the seeds are seeds
derived from a plant of the present invention.
[0208] The present invention also provides a container of over
about 10 kg, more preferably about 25 kg, and even more preferably
about 50 kg seeds where over about 10%, more preferably about 25%,
more preferably about 50%, and even more preferably about 75% or
more preferably about 90% of the seeds are seeds derived from a
plant of the present invention.
[0209] Any of the plants or parts thereof of the present invention
may be processed to produce a feed, meal, protein, or oil
preparation. A particularly preferred plant part for this purpose
is a seed. In a preferred embodiment the feed, meal, protein, or
oil preparation is designed for ruminant animals. Methods to
produce feed, meal, protein, and oil preparations are known in the
art. See, for example, U.S. Pat. Nos. 4,957,748; 5,100,679;
5,219,596; 5,936,069; 6,005,076; 6,146,669; and 6,156,227. In a
preferred embodiment, the protein preparation is a high protein
preparation. Such a high protein preparation preferably has a
protein content of greater than 5% w/v, more preferably 10% w/v,
and even more preferably 15% w/v.
[0210] In a further embodiment, meal of the present invention may
be blended with other meals. In a preferred embodiment, the meal
produced from plants of the present invention or generated by a
method of the present invention constitutes greater than about
0.5%, about 1%, about 5%, about 10%, about 25%, about 50%, about
75%, or about 90% by volume or weight of the meal component of any
product. In another embodiment, the meal preparation may be blended
and can constitute greater than about 10%, about 25%, about 35%,
about 50%, or about 75% of the blend by volume.
[0211] The present invention further provides a method of
up-regulating the expression of one or more CDK inhibitor-like
genes in a maize cell, a maize tissue, a maize organ, or a maize
plant. The method comprises contacting a maize cell, tissue, organ,
or plant with at least one nucleic acid construct comprising at
least one nucleotide sequence selected from the group consisting of
SEQ ID NO: 1 [ZmKRP1], a nucleotide sequence encoding the amino
acid sequence of SEQ ID NO: 2 [ZmKRP1], SEQ ID NO: 3 [ZmKRP2], a
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:
4 [ZmKRP2], SEQ ID NO: 7 [ZmKRP4], a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 8 [ZmKRP4], SEQ ID NO: 9
[ZmKRP5], a nucleotide sequence encoding the amino acid sequence of
SEQ ID NO: 10 [ZmKRP5], SEQ ID NO: 11 [ZmKRP6], and a nucleotide
sequence encoding the amino acid sequence of SEQ ID NO: 12
[ZmKRP6], wherein, when a cell, tissue, or organ is contacted with
the nucleic acid construct, the method further comprises generating
a plant from the cell, tissue, or organ, whereupon the expression
of one or more CDK inhibitor-like genes is up-regulated in a maize
cell, tissue, organ, or plant. The method can comprise contacting
the cell, tissue, organ, or plant with more than one nucleic acid
construct and each nucleic acid construct comprises a different
nucleotide sequence. The nucleic acid can comprise a promoter that
is preferentially expressed in the male reproductive tissue of
maize, wherein the promoter is operably linked to the at least one
nucleotide sequence. An example of such a promoter is the SILKY1
promoter of maize and others as described herein above.
Preferential expression in the male reproductive tissue of maize
can result in the generation of a male-sterile maize plant.
EXAMPLES
[0212] The present invention is described further in the context of
the following examples. These examples serve to illustrate further
the present invention and are not intended to limit the scope of
the invention.
Example 1
[0213] This example describes the identification of CDK inhibitors
from maize.
[0214] Initial tBLASTn searches (Altschul et al., Nuc. Acid Res.,
25:3389-3402 (1997)) of Monsanto's proprietary EST database were
performed using publicly available sequences of Arabidopsis CDK
inhibitor-like genes AtKRP1 ("At" indicates Arabidopsis thaliana;
KRP1 is as defined above; GenBank #AAC49698), AtKRP2 (GenBank
#CAB76424), AtKRP3 (GenBank #CAC41617), AtKRP4 (GenBank #CAC41618),
AtKRP5 (GenBank #CAC41619), AtKRP6 (GenBank #CAC41620), and AtKRP7
(GenBank #CAC41621). Additionally, the following conserved sequence
from the C-terminal region of the above polypeptides was used in a
tBLASTn search: (PTTAEIEDFFSEAEEQQQKQFIEKYNFDIVNDEPLEGRYEWVKLKP)
[SEQ ID NO: 59].
[0215] Resulting from the BLAST searches, seven maize sequence
clusters were identified as having homology to the above-mentioned
Arabidopsis CDK inhibitor-like genes. As used herein, "sequence
clusters" refer to a group of EST sequences with overlapping
regions of homology. Representative clones from each cluster were
full-length double-strand sequenced and were used to BLAST search
against Monsanto proprietary EST databases. An additional 2
clusters were identified from this second BLAST search and the
representative clones were full-length double-strand sequenced. DNA
sequence analysis indicated the presence of at least 9 different
CDK inhibitor-like clusters in maize. These 9 clusters and their
representative clones (named ZmKRP1 through ZmKRP9) are summarized
in Table 1.
TABLE-US-00001 TABLE 1 Maize CDK inhibitor-like clusters and
representative EST clones and ORF SEQ ID NO: ORF SEQ ID CLUSTER ID
SELECTED CLONE ID Description NO: ZEAMA-21JUN01-
LIB3732-047-Q1-N6-G6 ZmKRP1 1 CLUSTER89801_1 ZEAMA-04OCT00-
LIB3279-047-P1-K1-A7 ZmKRP2 3 CLUSTER67903_1 ZEAMA-21JUN01-
LIB3606-029-Q1-K6-B4 ZmKRP3 5 CLUSTER115879_1 ZEAMA-21JUN01-
LIB4759-013-R1-K1-B5 ZmKRP4 7 CLUSTER31652_2 ZEAMA-06JUN02-
LIB3898-003-Q1-N6-D10 ZmKRP5 9 CLUSTER620952_1 ZEAMA-21JUN01-
LIB143-038-Q1-E1-G6 ZmKRP6 11 CLUSTER301_55 ZEAMA-21JUN01-
700088048H1 ZmKRP7 13 CLUSTER301_41 ZEAMA-21JUN01-
LIB3587-222-Q1-K6-H9 ZmKRP8 15 CLUSTER301_46 ZEAMA-08NOV01-
LIB4574-012-Q1-K1-H11 ZmKRP9 17 CLUSTER33142_4
[0216] Coding regions and deduced amino acid sequences of the
representative clones of these 9 clusters are shown in the sequence
listing as SEQ ID NOs: 1-18. Full-length open reading frames (ORFs)
were identified for ZmKRP1, 2, 4, 5, and 6, while partial ORFs were
identified for ZmKRP3, 7, 8, and 9.
[0217] Protein sequence alignments indicated that maize KRPs share
small conserved regions with Arabidopsis ICK/KRPs, especially at
the C-terminal regions (FIG. 1). Additionally, some maize KRPs had
high sequence identity to each other. For example, ZmKRP6 and
ZmKRP7 were 90% identical, while ZmKRP1 and ZmKRP2 were 84%
identical. Other ZmKRP members shared only 16-50% identity.
[0218] Sequence alignments and percent identity calculations were
performed by the Clustal W Algorithm.
Example 2
[0219] This example describes the identification of CDK inhibitors
targeted for suppression in corn embryos.
[0220] The Monsanto proprietary corn cDNA database was queried
using DNA sequences from the maize KRP coding regions identified in
Example 1 (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17) using the
CDK BLASTn protocol. This was done to determine which sequences
were represented in cDNA libraries from developing kernel/embryo
tissues, as well as various non-kernel tissues. cDNA sequences
corresponding to ZmKRP1, ZmKRP2, ZmKRP4, ZmKRP6, ZmKRP7, and ZmKRP9
(SEQ ID NOs: 1, 2, 4, 6, 7, and 9) were identified in kernel or
embryo cDNA libraries. cDNA sequences corresponding to ZmKRP3,
ZmKRP5, and ZmKRP8 were found only in libraries generated from
non-kernel libraries. These results indicated that at least the
group of genes represented by the sequences found in the kernel or
embryo cDNA libraries needed to be targeted for suppression.
However, failure to identify cDNA sequences corresponding to a
given gene in kernel libraries does not preclude the possibility of
kernel expression, as a sequence may not be represented in a given
library. Because all 9 genes may be expressed in the embryo, all
were targeted for suppression using a dsRNA suppression
construct.
Example 3
[0221] This example describes the construction of vectors for the
tissue-specific suppression of the ZmKRP genes and the
transformation of corn plants with these vectors.
[0222] To suppress the 9 maize CDK inhibitor-like genes identified
in Example 1, a dsRNA strategy was used. To build the dsRNA
construct to suppress all 9 ZmKRP genes. (ZmKRP1-9), 200 base pair
fragments (F) were selected from the 3' end region of each gene (F1
to F7, SEQ ID NOs: 19 to 25), except for ZmKRP9, where 183 base
pairs were selected (SEQ ID NO: 26) and connected in a
polycistronic fashion. Since ZmKRP6 and ZmKRP7 are identical in
this 3' 200 base pair region, one fragment was used to target
suppression of both genes (F6). To make the final corn expression
construct, two intermediate constructs were first generated. The
construction of the 2 intermediate dsRNA constructs is shown
schematically in FIGS. 4 and 5.
[0223] Each intermediate dsRNA construct contained 4 of the 3'-end
fragments in both the sense and antisense orientation separated by
an intervening intron region. The first intermediate construct
contained the F1 through F4 sequences, and the second contained
sequences F5 through F8. The two stem-loop structures, with
respective promoters and 3' UTRs, were combined in one corn
expression vector (FIG. 2) and used for corn transformation using
the Agrobacterium transformation method described in Example
10.
[0224] To build the intermediate dsRNA construct containing the F1
to F4 sequences, the sense connections of F1 to F4, antisense
connections of F1 to F4, and ZmDnaK (gene encoding for HSP70)
intron fragment (SEQ ID NO: 27) (see e.g., U.S. Pat. No. 5,424,412;
Rochester et al., EMBO J., 5:451-458 (1986)) were individually
generated by PCR and assembled using a BamHI/BglII directional
cloning method (FIG. 4). The PCR methods followed standard
protocols set forth by the manufacturer (Roche Applied Science,
Indianapolis, Ind.), and adding 5% DMSO, unless otherwise noted.
The DMSO was added due to the high GC content of corn DNA.
[0225] The F1 fragment was PCR amplified from the DNA of the ZmKRP1
EST clone using primers P1-StuI (SEQ ID NO: 28) and P1-2R (SEQ ID
NO: 29). The primers are set forth in Table 2 and shown in FIG. 5.
The F1 fragment was then purified using standard methodology
well-known in the art. The F1-2 fragment was generated using PCR
amplification by using the purified F1 PCR product mixed with an
equivalent amount of the DNA of the ZmKRP2 EST clone as template,
and P1-StuI (SEQ ID NO: 28) and P2-3 (SEQ ID NO: 30) as primers.
The PCR product F1-2 was gel-purified using standard methodology.
Similarly, F3 and F4 were PCR connected using primers P4-Bgl (SEQ
ID NO: 31), P3-4 (SEQ ID NO: 32), and P5-Bam (SEQ ID NO: 33)
resulting in a PCR product F3-4. The F1-2 and F3-4 products were
subsequently PCR connected using primers P1-StuI, P2-3, and P5-Bam
(SEQ ID NO: 33). The resulting PCR product, F1-4, was purified and
cloned into a vector using the Topo-TA cloning system (Invitrogen,
Carlsbad, Calif.). The resulting clone, F14B3-sense, was
full-length double-strand sequenced. The F1-4 fragment was then PCR
amplified from the F14B3-sense clone using primers P5-Bgl (SEQ ID
NO: 34) and P1-SseBam (SEQ ID NO: 35) to generate the antisense
fragment. The resulting PCR product was again cloned using the
Topo-TA cloning system to form the F14B3-antisense clone. This
clone was confirmed by sequencing using standard methodology.
TABLE-US-00002 TABLE 2 Primers used in construction of ZmKRP dsRNA
vector PCR/CONNECTIONS PRIMERS SEQ ID NO F1 and F2 P1-StuI 28 P1-2R
29 P2-3 30 F3 and F4 P4-Bgl 31 P3-4 32 P5-Bam 33 F1-2 and F3-4
P1-StuI 28 P2-3 30 P5-Bam 33 F1-4 antisense P5-Bgl 34 P1-SseBam 35
ZmDnaK intron Pi-Bgl 36 Pi-Bam 37 F5 and F6 P5-Nco 38 P5-6 39 P6-7
40 F7 and F8 P8-Bgl 41 P7-8 42 P8-i 43 F5-6 and F7-8 P5-Nco 38 P6-7
40 P8-i 43 F5-8 and ZmDnaK intron P5-Nco 38 P8-i 43 Pi-Bam 37
[0226] The ZmDnaK-intron (see e.g., U.S. Pat. No. 5,424,412) was
PCR amplified from the plant expression vector pMON70091 which
contains a Z27 zein promoter (Lopes et al., Mol. Gen. Genet.,
247:603-613 (1995) and ZmDnaK intron using primers Pi-Bgl (SEQ ID
NO: 36) and Pi-Bam (SEQ ID NO: 37). The PCR product, DnaK-1, was
cloned using the Topo-TA cloning system. This clone was confirmed
by full-length double-strand sequencing. The F1-4 sense, ZmDnaK
intron, and F1-4 antisense fragments were assembled in a step-wise
fashion. Firstly, the ZmDnaK intron was excised from the DnaK-1
construct described above using BglII and BamHI. This intron was
then ligated to the BamHI site of F14B3-sense construct, such that
the BglII site of ZmDnaK-intron/BamHI-BglII ligated to the BamHI
site (introduced by primer P5-Bam) at the 3' end of F1-4. This
ligation reaction yielded the construct F14In2. Secondly, the F1-4
fragment was excised from clone F14B3-antisense with BglII and
BamHI, and the F1-4/BglII-BamHI fragment was ligated to the BamHI
site of F14In2, such that the BglII site of F1-4/BglII-BamHI
ligated to the BamHI site (introduced by primer Pi-Bam) at the 3'
end of the ZmDnaK intron. This ligation reaction yielded the
construct F142c1.
[0227] Finally, the whole DNA segment containing the F1-4 sense;
ZmDnaK intron; F1-4 antisense fragment was excised from clone
F142c1 using StuI and Sse83871 and cloned into pMON80611. pMON80611
is a binary vector for Agrobacterium transformation of plants. It
contains left and right borders for T-DNA transfer, a plant
selectable marker cassette consisting of enhanced 35S
promoter::glyphosate resistance marker gene::nos 3' UTR (U.S. Pat.
No. 5,627,061) and plant expression cassette sequences which
include an oleosin L3 promoter (U.S. Pat. No. 6,433,252) and a
maize globulin 3' UTR (see, for example, Belanger and Kriz, Genet.,
129:863-872, (1991)) with cloning sites available for expression of
a gene of interest. The resulting expression cassette was named
WWZHEN03.0024.
[0228] The intermediate dsRNA construct containing the F5 to F8
fragments was then generated. The sense connections of F5 to F8,
antisense connections of F5 to F8, and ZmDnaK intron (SEQ ID NO:
27) were individually generated by PCR and assembled using either
PCR or the BamHI/BglII directional cloning method as described
above and shown in FIG. 5. Primers used in those PCR connections
were listed in Table 2 and FIG. 5. The assembled segment comprising
F5-8 sense; ZmDnaK intron; F5-8 antisense was then cloned to vector
pMON80611 to generate the second expression cassette WWZHEN03.0023.
To combine WWZHEN03.0023 and WWZHEN03.0024, the 4528 bp AscI-FseI
segment from WWZHEN03.0023 that comprising F5-8 sense; ZmDnaK
intron; F5-8 antisense was gel purified (Qiagen Inc., Valencia,
Calif.) and ligated to WWZHEN03.0024 digested with MluI and FseI to
generate the final corn transformation construct pMON71270 (FIG.
2).
[0229] A Perl promoter-driven ZmKRP suppression construct was made
from the two intermediate dsRNA contructs WWZHEN03.0023 and
WWZHDN03.0024 (above). First, the segment comprising F1-4 sense;
ZmDnaK intron; F1-4 antisense was excised from WWZHDN03.0024 by
SfiI and Sse83871 digestion, and then cloned to the SfiI and
Sse83871 sites of vector pMON68290 which contained Perl to generate
WWZHEN03.0041. Similarly, the segment comprising F5-8 sense; ZmDnaK
intron; F5-8 antisense was excised from WWZHEN03.0023 by SfiI and
Sse83871 digestion, and then cloned to the SfiI and Sse83871 sites
of vector pMON68290 which contained Perl to generate WWZHEN03.0042.
To combine WWZHEN03.0041 and WWZHEN03.0042, the 4481 bp AscI
segment from WWZHEN03.0042, comprising F5-8 sense; ZmDnaK intron;
F5-8 antisense, was gel purified (Qiagen Inc., Valencia, Calif.)
and ligated to WWZHEN03.0041 digested with MluI to generate the
final corn transformation construct pMON71279 (FIG. 3).
Example 4
[0230] This example sets forth the construction of plant
transformation vectors for the suppression of one or more ZmKRP
genes in corn.
[0231] Additional seed-specific suppression constructs are
generated using alternative size fragments, ranging from 50 base
pairs to the full-length gene, and either alone or in various
combinations. By way of example, a dsRNA construct using a 200 base
pair fragment of ZmKRP1 operably linked to the corn L3 (oleosin)
promoter is generated. The F1 fragment and the ZmDnaK-intron are
generated as described above in Example 3. The two fragments are
ligated to generate an intermediate F1; ZmDnaK-intron fragment. An
aliquot of isolated and purified F1 fragment is then PCR amplified
to generate F1-antisense. The F1-antisense is then ligated to the
intermediate F1; ZmDnaK-intron fragment to generate a dsRNA
construct F1; ZmDnaK-intron; F1-antisense. The construct is then
cloned into a vector downstream of the corn L3 (oleosin) promoter.
Alternatively the construct is cloned into a vector downstream of a
promoter such as P-Zm.CEP1, P-Zm.CPC214, P-Zm.CPC214tr1,
P-Zm.CPC214tr2, P-Os.CPC214, and P-Hv.PER1 as well as other
embryo-preferred promoters known to those skilled in the art.
Similar constructs are generated with ZmKRP2, ZmKRP3, ZmKRP4,
ZmKRP5, ZmKRP6, ZmKRP7, ZmKRP8, and ZmKRP9. Transgenic corn plants
are produced. Transgenic seed is analyzed from these constructs to
identify alterations in germ mass, kernel mass, cellular
proliferation, whole kernel oil levels, whole kernel protein levels
(% dry weight), whole kernel amino acid composition, free amino
acid levels, and micronutrient composition, as set forth in the
examples that follow. The ZmKRP gene fragments used in dsRNA
constructs, which result in alterations in any of the
abovementioned properties, are then combined, depending on the
desired phenotype of the transformed plant, in a single
polycistronic dsRNA construct, which is then transformed into corn
using Agrobacterium transformation; transgenic plants are generated
as described in Example 10.
Example 5
[0232] This example describes how the expression of endogenous CDK
inhibitor-like genes in a plant can be suppressed by using
antisense technology.
[0233] The level of ZmKRP1 generated in a cell of a corn plant can
be decreased by introducing into the genome a transgene that
expresses an antisense RNA of the ZmKRP1 polypeptide. For example,
the ZmKRP1 cDNA, or a fragment thereof at least 100 bp in length,
is cloned into the plant expression vector pMON80611 in the
antisense orientation, which drives expression under control of the
L3 promoter and contains the globulin 3' UTR (Belanger and Kriz,
Genet., 129:863-872 (1991)). The resulting vector is transformed
into the genome of a corn plant as described in Example 10.
Multiple copies of the antisense gene can be introduced into a
genome. Other CDK inhibitor-like genes, for instance ZmKRP2, can be
made into antisense constructs and expressed separately or in
combinations, as described above for ZmKRP1.
Example 6
[0234] This example describes how the expression of endogenous CDK
inhibitor-like genes in a plant can be suppressed by using ribozyme
technology.
[0235] The level of ZmKRP1 mRNA generated in a cell of a corn plant
can be decreased by introducing into the genome a transgene that
expresses a ribozyme targeted against an mRNA coding for a ZmKRP1
polypeptide. The ribozyme construct is generated by cloning at
least 24 bp of ZmKRP1 cDNA with a ribozyme sequence added adjacent
to the target AUG of the endogenous gene (Merlo et al., Plant Cell,
10(10):1603-1622 (1998)) into pMON80611, which drives expression
from the L3 promoter and contains the globulin 3' UTR (Belanger and
Kriz, Genet., 129:863-872 (1991)). The resulting construct is
transformed into a corn plant as described in Example 10. Multiple
copies of the ribozyme-containing ZmKRP genes can be introduced
into a genome as described in Example 10. Ribozyme-containing genes
targeted against a combination of other ZmKRP polypeptides (as
described herein above) can be introduced into a genome as
described above for ZmKRP1, such that multiple ZmKRP genes are
simultaneously suppressed.
Example 7
[0236] This example describes how the expression of endogenous CDK
inhibitor genes in a plant can be suppressed by using homologous
recombination technology.
[0237] The level of ZmKRP1 generated in a cell of a corn plant can
be decreased by a gene replacement method via homologous
recombination. In this method, the endogenous ZmKRP1 gene is
replaced by a mutagenized ZmKRP1 gene. The mutagenized gene codes
for a ZmKRP1 that is less efficient, or completely deficient, in
inhibition of CDK than the one encoded by the endogenous, replaced
gene. The ZmKRP1-producing gene is mutagenized by one or more
nucleotide deletions, insertions, duplications or replacements. The
mutagenized gene is fused to a selectable marker gene and
transformed into a cell of a corn plant as described in Example 10.
Homologous recombination events that may result in gene replacement
are selected on the basis of the selectable marker gene (Puchta et
al., Trends Plant Sci., 1:340 (1996); Kempin et al., Nature,
389:802 (1997)). Gene replacements are confirmed by Southern blot
analysis or PCR and DNA sequencing.
[0238] In the same manner as described above, other CDK inhibitor
genes can be suppressed, independently or in combinations.
Example 8
[0239] This example describes how the expression of endogenous CDK
inhibitor genes in a plant can be suppressed by using sense
suppression technology.
[0240] The level of ZmKRP1 polypeptide generated in a cell of a
plant is decreased by sense suppression. For example, the cDNA
coding for an ZmKRP1 polypeptide is fused to upstream (5') such as
Perl, and/or downstream (3') transcriptional or translational
regulatory sequences and the chimeric gene is cloned into an
appropriate transformation vector that carries a selectable marker
gene and the vector is transformed into a cell of a corn plant as
described in Example 10. Transformants are selected on the basis of
the presence of a marker gene (for instance glyphosate resistance).
Transformants are confirmed by Southern blot analysis of the DNA
from putative transformants. Most of the transgenic organisms that
will be derived from such experimentations will express the
transgene without affecting endogenous expression. However, in a
few cases, both of the transgene and the endogenous gene will be
suppressed. To identify these sense-suppressed plants, RNA or
protein extracts from at least 60 transgenic plants will be
analyzed for the presence of the ZmKRP mRNA or polypeptide,
respectively. Other CDK inhibitor-like genes can be used, alone or
in various combinations.
Example 9
[0241] This example sets forth one method of generating male
sterile maize plants by expressing maize CDK inhibitors in male
reproductive tissue.
[0242] The corn SILKY1 promoter is operably linked to ZmKRP1,
ZmKRP2, ZmKRP4, ZmKRP5, and ZmKRP6 in separate expression
constructs containing the nos 3' UTR (Bevan et al., Nucl. Acid
Res., 11:369 (1983)) (other possibilities exist) and a glyphosate
resistance selectable marker. (See, U.S. Pat. No. 5,627,061). One
or more of the resulting expression cassettes is/are transformed
into corn using the Agrobacterium transformation protocol set forth
in Example 10 and transgenic plants are generated.
[0243] Male sterility is determined by performing pollen
germination assays (Walden, D. B. (1994), In vitro Pollen
Germination (pp 723-724), The Maize Handbook, Freeling, M. and
Walbot, V. eds, Springer-Verlag, New York, Inc.), if pollen is
present. Additionally, pollen viability is assayed by the ability
of the transgenic pollen to fertilize ovules of non-transgenic
plants and to produce viable seed harboring the transgene. Sterile
transgenic plants are maintained by fertilizing the transgenic
plants with non-transgenic donor pollen.
Example 10
[0244] This example describes the transformation of maize plants
with constructs containing maize CDK inhibitor-like sequences.
[0245] The transformation vectors described above are used to
transform maize plants using the following procedure. Maize plants
are grown in a greenhouse under standard practices. Controlled
pollinations are made. Agrobacterium ABI containing a vector in
glycerol stock is streaked out on solid LB medium supplemented with
antibiotics kanamycin (50 mg/L), spectinomycin (50 mg/L),
streptomycin (50 mg/L), and chloramphenicol (25 mg/L) and incubated
at 28.degree. C. for 2 days. Two days before Agrobacterium
inoculation of the maize immature embryos, one colony or a small
loop of Agrobacterium from the Agrobacterium plate is picked up and
inoculated into 25 mL of liquid LB medium supplemented with 100
mg/L of spectinomycin and 50 mg/L of kanamycin in a 250-mL flask.
The flask is placed on a shaker at approximately 150 rpm and
26.degree. C. overnight. The Agrobacterium culture is then diluted
(1 to 5) in the same liquid medium and put back on the shaker.
Several hours later, one day before inoculation, the Agrobacterium
cells are spun down at 3500 rpm for 15 min. The bacterium cell
pellet is re-suspended in induction broth with 200 .mu.M of
acetosyringone and 50 mg/L spectinomycin and 25 mg/L kanamycin and
the cell density was adjusted to 0.2 at O.D..sub.660. The bacterium
cell culture (50 mL in each 250-mL flask) is then put back on the
shaker and grown overnight. On the morning of inoculation day, the
bacterium cells are spun down and washed with liquid 1/2MS VI
medium (Table 3) supplemented with 200 .mu.M of acetosyringone.
After one more spinning, the bacterium cell pellet is re-suspended
in 1/2 MS PL medium (Table 3) with 200 .mu.M of acetosyringone
(Table 3) and the cell density was adjusted to 1.0 at O.D.sub.660
for inoculation.
[0246] Reagents are commercially available and can be purchased
from a number of suppliers (see, for example, Sigma Chemical Co.,
St. Louis, Mo.).
TABLE-US-00003 TABLE 3 Media used.sup.1. Co- 1/2 MS 1/2 MS culture
Induction Component VI PL medium MS MSW50 MS/6B A MSOD MS salts
68.5 g/l 68.5 g/l 2.2 g/l 4.4 g/l 4.4 g/l 4.4 g/l 4.4 g/l Sucrose
20 g/l 68.6 g/l 20 g/l 30 g/l 30 g/l 30 g/l -- Maltose -- -- -- --
-- -- 20 g/l Glucose 10 g/l 36 g/l 10 g/l -- -- -- 10 g/l l-Proline
115 mg/l 115 mg/l 115 mg/l 1.36 g/l 1.38 g/l 1.36 g/l -- Casamino
-- -- -- 50 mg/l 500 mg/l 50 mg/l -- Acids Glycine 2 mg/l 2 mg/l 2
mg/l -- 2 mg/l -- -- l-Asparagine -- -- -- -- -- -- 150 mg/l
myo-Inositol 100 mg/l 100 mg/l 100 mg/l -- 100 mg/l -- 100 mg/l
Nicotinic 0.5 mg/l 0.5 mg/l 0.5 mg/l 1.3 mg/l 0.5 mg/l 1.3 mg/l 1.3
mg/l Acid Pyridoxine.cndot.HCl 0.5 mg/l 0.5 mg/l 0.5 mg/l 0.25 mg/l
0.5 mg/l 0.25 mg/l 0.25 mg/l Thiamine.cndot.HCl 0.1 mg/l 0.1 mg/l
0.6 mg/l 0.25 mg/l 0.6 mg/l 0.25 mg/l 0.25 mg/l Ca -- -- -- 0.25
mg/l -- 0.25 mg/l 0.25 mg/l Pantothenate -- -- 3 mg/l 0.5 mg/l 0.5
mg/l -- -- 2,4-D -- -- -- 2.2 mg/l -- -- -- Picloram Silver Nitrate
-- -- 1.7 mg/l 1.7 mg/l -- -- -- BAP -- -- -- -- -- 3.5 mg/l
.sup.1Media 1/2 MSVI and 1/2 MSPL were used as liquid. Co-culture
medium was solidified with 5.5 mg/l low EEO agarose. All other
media were solidified with 7 g/l Phytagar for NPTII selection and
with 3 g/l phytagel for glyphosate selection.
[0247] An elite corn line (LH244) is used for transformation in
connection with this invention. Ears containing immature embryos
are harvested approximately 10 days after pollination and kept
refrigerated at 4.degree. C. until use (up to 5 days post-harvest).
The preferred embryo size for this method of transformation is
.about.1.0-2.0 mm. This size is usually achieved 10 days after
pollination inside the green house with the growth conditions of an
average temperature of 87.degree. F., day length of 14 hours with
supplemental lighting supplied by GE 1000 Watt High Pressure Sodium
lamps.
[0248] Immature embryos are isolated from surface sterilized ears
and directly dropped into the prepared Agrobacterium cell
suspension in 1.5-mL microcentrifuge tube. The isolation lasts
continuously for 15 min. The tube is then set aside for 5 min,
which made the inoculation time for individual embryos from 5 to 20
min. After Agrobacterium cell suspension is removed using a fine
tipped sterile transfer pipette, the immature embryos are
transferred onto the co-culture medium (Table 3). The embryos are
placed on the medium with the scutellum side facing up. The embryos
are cultured in a dark incubator (23.degree. C.) for approximately
24 h.
[0249] The embryos are then transferred onto a modified MS medium
(MSW50, Table 3) supplemented with 0.1 or 0.25 mM glyphosate and
250 mg/L carbenicillin to inhibit Agrobacterium in Petri dishes
(100 mm.times.25 mm). The cultures are incubated in a dark culture
room at 27.degree. C. for 2-3 weeks. All the callus pieces are then
transferred individually onto the first regeneration medium
(MS/6BA, Table 3) supplemented with the same levels of glyphosate.
The cultures are grown on this medium and in a culture room with
16-h light/8-h dark photoperiod and 27.degree. C. for 5-7 days.
They are then transferred onto the second regeneration medium
(MSOD, Table 3) in petri dish (100 mm.times.25 mm) for
approximately 2 weeks. All the callus pieces with regenerating
shoots and living tissue are transferred onto the same medium
contained in phytatrays for shoots to grow further before being
moved to soil. It takes 2-4 weeks. The regeneration media (MS6BA
and MSOD) are all supplemented with 250 mg/L carbenicillin and 0.1
or 0.25 mM glyphosate.
[0250] These developing plantlets are then transferred to soil,
hardened off in a growth chamber at 27.degree. C., 80% humidity,
and low light intensity for approximately 1 week, and then
transferred to a greenhouse and grown under standard greenhouse
conditions.
[0251] The greenhouse-grown plants are then analyzed for expression
levels as well as oil and protein levels.
Example 11
[0252] This example describes expression analysis of transgenic
plants.
[0253] RNA expression analysis is used to verify misexpression or
suppression of CDK inhibitors in transgenic plants. As used in this
application, "misexpression" means spatial or temporal expression
other than endogenous expression, e.g. overexpression and ectopic
expression. RNA is extracted by a standard protocol (Wadsworth et
al., Analytical Biochemistry, 172(1):279-283 (1988)). To determine
the misexpression or suppression of ZmKRP genes in corn embryo
tissues, TaqMan analysis is performed using the TaqMan One-Step
RT-PCR Master Mix Reagents Kit and Protocol (#4310299 rev. C)
(Applied Biosystems, Foster City Calif.). cDNA is synthesized
(SMART PCR cDNA Synthesis Kit, #K1052-1, Clonetech Laboratories,
Inc.) and standard PCR using gene-specific primers and agarose-gel
electrophoresis to visualize PCR products.
Example 12
[0254] This example provides the analytical procedures to determine
oil and protein content, mass differences, amino acid composition,
free amino acid levels, and micronutrient content of transgenic
maize plants.
[0255] Oil levels (on a mass basis and as a percent of tissue
weight) of first generation single corn kernels and dissected germ
and endosperm are determined by low-resolution .sup.1H nuclear
magnetic resonance (NMR) (Tiwari et al., JAOCS, 51:104-109 (1974);
or Rubel, JAOCS, 71:1057-1062 (1994)), whereby NMR relaxation times
of single kernel samples are measured, and oil levels are
calculated based on regression analysis using a standard curve
generated from analysis of corn kernels with varying oil levels as
determined gravimetrically following accelerated solvent
extraction. One-way analysis of variance and the Student's T-test
(JMP, version 4.04, SAS Institute Inc., Cary, N.C., USA) are
performed to identify significant differences between transgenic
and non-transgenic kernels as determined by transgene-specific
PCR.
[0256] Oil levels and protein levels in second generation seed are
determined by NIT spectroscopy, whereby NIT spectra of pooled seed
samples harvested from individual plants are measured, and oil and
protein levels are calculated based on regression analysis using a
standard curve generated from analysis of corn kernels with varying
oil or protein levels, as determined gravimetrically following
accelerated solvent extraction or elemental (% N) analysis,
respectively. One-way analysis of variance and the Student's T-test
are performed to identify significant differences in oil (% kernel
weight) and protein (% kernel weight) between seed from marker
positive and marker negative plants.
[0257] The levels of free amino acids are analyzed from each of the
transgenic events using the following procedure. Seeds from each of
the transgenic plants are crushed individually into a fine powder
and approximately 50 mg of the resulting powder is transferred to a
pre-weighed centrifuge tube. The exact sample weight is recorded
and 1.0 ml of 5% trichloroacetic acid is added to each sample tube.
The samples are mixed at room temperature by vortex and then
centrifuged for 15 minutes at 14,000 rpm on an Eppendorf
microcentrifuge (Model 5415C, Brinkmann Instrument, Westbury,
N.Y.). An aliquot of the supernatant is removed and analyzed by
HPLC (Agilent 1100) using the procedure set forth in Agilent
Technical Publication "Amino Acid Analysis Using the Zorbax
Eclipse-AAA Columns and the Agilent 1100 HPLC," Mar. 17, 2000.
[0258] Quantitative determination of total amino acids from corn is
performed by the following method. Kernels are ground and
approximately 60 mg of the resulting meal is acid-hydrolyzed using
6 N HCl under reflux at 100.degree. C. for 24 hrs. Samples are
dried and reconstituted in 0.1 N HCl followed by precolumn
derivatization with .alpha.-phthalaldehyde (OPA0 for HPLC analysis.
The amino acids are separated by a reverse-phase Zorbax Eclipse
XDB-C18 HPLC column on an Agilent 1100 HPLC (Agilent, Palo Alto,
Calif.). The amino acids are detected by fluorescence. Cysteine,
proline, asparagine, glutamine, and tryptophan are not included in
this amino acid screen (Henderson et al., "Rapid, Accurate,
Sensitive and Reproducible HPLC Analysis of Amino acids, Amino Acid
Analysis Using Zorbax Eclipse-AAA Columns and the Agilent 1100
HPLC," Agilent Publication (2000); see, also, "Measurement of
Acid-Stable Amino Acids," AACC Method 07-01 (American Association
of Cereal Chemists, Approved Methods, 9th edition (LCCC#
95-75308)). Total tryptophan is measured in corn kernels using an
alkaline hydrolysis method as described (Approved Methods of the
American Association of Cereal Chemists--10.sup.th edition, AACC
ed, (2000) 07-20 Measurement of Tryptophan--Alakline
Hydrolysis).
[0259] Tocopherol and tocotrienol levels in seeds are assayed by
methods well-known in the art. Briefly, 10 mg of seed tissue are
added to 1 g of microbeads (Biospec Product Inc, Barlesville,
Okla.) in a sterile microfuge tube to which 500 .mu.l 1% pyrogallol
(Sigma Chemical Co., St. Louis, Mo.)/ethanol have been added. The
mixture is shaken for 3 minutes in a mini Beadbeater (Biospec) on
"fast" speed, then filtered through a 0.2 .mu.m filter into an
autosampler tube. The filtered extracts are analyzed by HPLC using
a Zorbax silica HPLC column (4.6 mm.times.250 mm) with a
fluorescent detection, an excitation at 290 nm, an emission at 336
nm, and bandpass and slits. Solvent composition and running
conditions are as listed below with solvent A as hexane and solvent
B as methyl-t-butyl ether. The injection volume is 20 .mu.l, the
flow rate is 1.5 ml/minute and the run time is 12 minutes at
40.degree. C. The solvent gradient is 90% solvent A, 10% solvent B
for 10 minutes; 25% solvent A, 75% solvent B for 11 minutes; and
90% solvent A, 10% solvent B for 12 minutes. Tocopherol standards
in 1% pyrogallol/ethanol are run for comparison
(.alpha.-tocopherol, .gamma.-tocopherol, .beta.-tocopherol,
.delta.-tocopherol, and tocopherol (tocol)). Standard curves for
alpha, beta, delta, and gamma tocopherol are calculated using
Chemstation software (Hewlett Packard). Tocotrienol standards in 1%
pyrogallol/ethanol are run for comparison (.alpha.-tocotrienol,
.gamma.-tocotrienol, .beta.-tocotrienol, .delta.-tocotrienol).
Standard curves for .alpha.-, .beta.-, .delta.-, and
.gamma.-tocotrienol are calculated using Chemstation software
(Hewlett Packard).
[0260] Carotenoid levels within transgenic corn kernels are
determined by a standard protocol (Craft, Meth. Enzymol.,
213:185-205 (1992)). Plastiquinols and phylloquinones are
determined by standard protocols (Threlfall et al., Methods in
Enzymology, XVIII, part C, 369-396 (1971); and Ramadan et al., Eur.
Food Res. Technol., 214(6):521-527 (2002)).
[0261] Results from events containing pMON71270 are shown in Table
4.
TABLE-US-00004 TABLE 4 pMON71270 1st Generation Single Kernel
Analysis Positive GOI Negative GOI Pedigree n Mean LSD n Mean LSD
Prob > F Signif Delta LH244/ZM_S86489 Kernel Oil (%) 13 2.60
0.43 9 3.70 0.52 0.0001 0.001 -1.10 Germ Weight (g) 13 0.0191
0.00326 9 0.0212 0.00 0.2529 -0.0020 Germ Oil (%) 13 20.81 3.69 9
27.58 4.44 0.0025 0.01 -6.77 Germ Weight (%) 13 11.27 1.25 9 11.54
1.51 0.6894 -0.27 Kernel Weight (g) 13 0.1889 0.0174 9 0.2030
0.0209 0.1410 -0.0141 Kernel Oil (mg) 13 4.88 1.16 9 7.59 1.39
0.0003 0.001 -2.71 Germ Oil (mg) 13 3.95 0.97 9 5.74 1.16 0.0023
0.01 -1.79 ZM_S86363/LH244 Kernel Oil (%) 13 4.03 0.41 9 3.79 0.49
0.2772 0.24 Germ Weight (g) 13 0.0164 0.0026 9 0.0134 0.0031 0.9927
0.0030 Germ Oil (%) 13 31.17 1.91 9 30.65 2.30 0.6169 0.52 Germ
Weight (%) 13 12.51 1.25 9 12.09 1.51 0.5355 0.42 Kernel Weight (g)
13 0.1516 0.0223 9 0.1542 0.0268 0.8255 -0.0026 Kernel Oil (mg) 13
6.10 1.16 9 5.86 1.39 0.6997 0.24 Germ Oil (mg) 13 5.14 1.00 9 5.11
1.20 0.9554 0.03 ZM_S86474/LH244 Kernel Oil (%) 11 2.92 0.62 13
2.87 0.57 0.8689 0.05 Germ Weight (g) 11 0.0131 0.0060 13 0.0151
0.0055 0.4828 -0.0020 Germ Oil (%) 11 27.60 4.40 13 25.70 4.05
0.3613 1.90 Germ Weight (%) 11 9.24 2.99 13 10.56 2.75 0.3515 -1.32
Kernel Weight (g) 11 0.1554 0.0283 13 0.1581 0.0261 0.8346 -0.0028
Kernel Oil (mg) 11 4.70 1.52 13 4.67 1.40 0.9671 0.03 Germ Oil (mg)
11 3.61 1.35 13 3.65 1.25 0.9503 -0.04 ZM_S86481/LH244 Kernel Oil
(%) 10 3.98 0.21 13 3.69 0.19 0.0067 0.01 0.29 Germ Weight (g) 10
0.0188 0.0036 13 0.0174 0.0032 0.3901 0.0014 Germ Oil (%) 10 31.10
4.96 13 32.71 4.35 0.4794 -1.61 Germ Weight (%) 10 10.83 1.79 13
10.58 1.57 0.7637 0.25 Kernel Weight (g) 10 0.1916 0.0157 13 0.1850
0.0137 0.3640 0.0066 Kernel Oil (mg) 10 7.63 0.74 13 6.83 0.65
0.0254 0.05 0.80 Germ Oil (mg) 10 5.88 1.09 13 5.53 0.96 0.4867
0.35 ZM_S86483/LH244 Kernel Oil (%) 1 3.52 1.28 23 4.06 0.27 0.2410
-0.54 Germ Weight (g) 1 0.0104 0.0088 23 0.0137 0.0018 0.2920
-0.0033 Germ Oil (%) 1 21.15 8.38 23 31.42 1.75 0.0019 0.01 -10.27
Germ Weight (%) 1 11.11 3.55 23 11.94 0.74 0.5101 -0.83 Kernel
Weight (g) 1 0.1025 0.0719 23 0.1286 0.0150 0.3087 -0.0261 Kernel
Oil (mg) 1 3.61 3.02 23 5.21 0.63 0.1434 -1.60 Germ Oil (mg) 1 2.19
2.70 23 4.30 0.56 0.0352 0.05 -2.11 ZM_S86484/LH244 Kernel Oil (%)
13 3.85 0.23 11 3.75 0.25 0.4003 0.10 Germ Weight (g) 13 0.0186
0.0022 11 0.0187 0.0024 0.9640 0.0000 Germ Oil (%) 13 29.33 1.56 11
29.46 1.70 0.8678 -0.13 Germ Weight (%) 13 11.76 0.73 11 11.64 0.80
0.7358 0.13 Kernel Weight (g) 13 0.1769 0.0134 11 0.1786 0.0146
0.8060 -0.0017 Kernel Oil (mg) 13 6.82 0.67 11 6.70 0.73 0.7371
0.12 Germ Oil (mg) 13 5.47 0.67 11 5.48 0.72 0.9811 -0.01
ZM_S86486/LH244 Kernel Oil (%) 11 4.15 0.26 13 4.10 0.24 0.6438
0.06 Germ Weight (g) 11 0.0159 0.0024 13 0.0152 0.0022 0.5029
0.0007 Germ Oil (%) 11 32.65 2.06 13 32.05 1.89 0.5357 0.60 Germ
Weight (%) 11 10.64 1.25 13 10.37 1.15 0.6481 0.27 Kernel Weight
(g) 11 0.1657 0.0142 13 0.1630 0.0130 0.6841 0.0027 Kernel Oil (mg)
11 6.87 0.50 13 6.64 0.46 0.3396 0.23 Germ Oil (mg) 11 5.19 0.69 13
4.83 0.64 0.2764 0.36 ZM_S86487/LH244 Kernel Oil (%) 10 3.93 0.25
14 3.76 0.21 0.1476 0.17 Germ Weight (g) 10 0.0188 0.00266 14
0.0167 0.0023 0.0925 0.1 0.0021 Germ Oil (%) 10 32.54 2.42 14 32.35
2.04 0.8614 0.19 Germ Weight (%) 10 10.80 1.39 14 9.72 1.18 0.0991
0.1 1.07 Kernel Weight (g) 10 0.1919 0.0127 14 0.1905 0.0108 0.8070
0.0014 Kernel Oil (mg) 10 7.54 0.72 14 7.16 0.61 0.2471 0.38 Germ
Oil (mg) 10 6.10 0.90 14 5.38 0.76 0.0884 0.1 0.72 ZM_S86493/LH244
Kernel Oil (%) 9 3.66 0.69 15 3.94 0.53 0.3525 -0.28 Germ Weight
(g) 9 0.0177 0.0041 15 0.0161 0.0031 0.3593 0.0016 Germ Oil (%) 9
28.30 3.07 15 30.98 2.38 0.0548 0.1 -2.68 Germ Weight (%) 9 10.61
1.99 15 10.52 1.54 0.9196 0.09 Kernel Weight (g) 9 0.1813 0.0207 15
0.1699 0.0160 0.2153 0.0114 Kernel Oil (mg) 9 6.80 1.59 15 6.73
1.24 0.9196 0.07 Germ Oil (mg) 9 5.21 1.29 15 4.98 1.00 0.6775 0.23
ZM_S86521/LH244 Kernel Oil (%) 18 3.58 0.20 5 3.83 0.38 0.0965 0.1
-0.25 Germ Weight (g) 18 0.0134 0.0021 5 0.0155 0.0040 0.1935
-0.0021 Germ Oil (%) 18 30.82 1.91 5 28.40 3.63 0.0980 0.1 2.42
Germ Weight (%) 18 11.11 1.06 5 11.45 2.00 0.6653 -0.34 Kernel
Weight (g) 18 0.1374 0.0163 5 0.1430 0.0309 0.6382 -0.0057 Kernel
Oil (mg) 18 4.93 0.66 5 5.42 1.25 0.3195 -0.49 Germ Oil (mg) 18
4.09 0.57 5 4.40 1.08 0.4592 -0.31 ZM_S86533/LH244 Kernel Oil (%)
10 4.04 0.49 14 4.09 0.41 0.8145 -0.05 Germ Weight (g) 10 0.0196
0.0033 14 0.0183 0.0028 0.4218 0.0012 Germ Oil (%) 10 29.10 2.03 14
29.79 1.71 0.4554 -0.69 Germ Weight (%) 10 12.56 1.44 14 12.10 1.22
0.4821 0.46 Kernel Weight (g) 10 0.1732 0.0210 14 0.1684 0.0177
0.6203 0.0047 Kernel Oil (mg) 10 6.99 1.25 14 6.92 1.06 0.9001 0.07
Germ Oil (mg) 10 5.69 1.05 14 5.47 0.89 0.6410 0.22 ZM_S86541/LH244
Kernel Oil (%) 7 3.73 0.26 17 3.80 0.17 0.5521 -0.06 Germ Weight
(g) 7 0.0155 0.0044 17 0.0162 0.0028 0.6980 -0.0007 Germ Oil (%) 7
33.46 7.10 17 29.43 4.56 0.1752 4.03 Germ Weight (%) 7 11.72 2.37
17 11.38 1.52 0.7209 0.35 Kernel Weight (g) 7 0.1556 0.0279 17
0.1619 0.0179 0.5802 -0.0064 Kernel Oil (mg) 7 5.79 1.06 17 6.15
0.68 0.4167 -0.36 Germ Oil (mg) 7 4.86 1.19 17 4.78 0.76 0.8680
0.08 ZM_S86544/LH244 Kernel Oil (%) 13 3.89 0.21 10 4.01 0.24
0.2788 -0.12 Germ Weight (g) 13 0.0179 0.0028 10 0.0178 0.0032
0.9600 0.0001 Germ Oil (%) 13 30.41 2.18 10 31.53 2.49 0.3305 -1.12
Germ Weight (%) 13 11.52 1.00 10 10.90 1.14 0.2452 0.62 Kernel
Weight (g) 13 0.1735 0.0225 10 0.1804 0.0256 0.5562 -0.0069 Kernel
Oil (mg) 13 6.75 1.01 10 7.25 1.15 0.3401 -0.51 Germ Oil (mg) 13
5.41 0.81 10 5.56 0.93 0.7318 -0.15 ZM_S86548/LH244 Kernel Oil (%)
11 4.94 1.18 13 4.09 1.09 0.1344 0.85 Germ Weight (g) 11 0.0170
0.0027 13 0.0172 0.0024 0.8392 -0.0003 Germ Oil (%) 11 34.90 2.21
13 31.24 2.03 0.0017 0.01 3.66 Germ Weight (%) 11 12.54 2.70 13
10.70 2.48 0.1549 1.84 Kernel Weight (g) 11 0.1619 0.0261 13 0.1824
0.0240 0.1043 -0.0204 Kernel Oil (mg) 11 7.38 0.81 13 7.46 0.74
0.8349 -0.08 Germ Oil (mg) 11 5.89 0.90 13 5.38 0.83 0.2321 0.52
ZM_S86551/LH244 Kernel Oil (%) 9 3.76 0.25 15 3.88 0.19 0.2731
-0.12 Germ Weight (g) 9 0.0223 0.0031 15 0.0213 0.0024 0.4592
0.0010 Germ Oil (%) 9 30.44 1.63 15 31.37 1.26 0.1969 -0.94 Germ
Weight (%) 9 11.32 1.18 15 10.80 0.91 0.3228 0.51 Kernel Weight (g)
9 0.2200 0.0180 15 0.2200 0.0139 0.9957 0.0000 Kernel Oil (mg) 9
8.26 0.86 15 8.53 0.67 0.4798 -0.27 Germ Oil (mg) 9 6.78 1.04 15
6.71 0.80 0.8741 0.07 ZM_S86571/LH244 Kernel Oil (%) 17 3.81 0.20 7
3.83 0.31 0.8670 -0.02 Germ Weight (g) 17 0.0211 0.0025 7 0.0216
0.0039 0.7492 -0.0005 Germ Oil (%) 17 29.76 2.16 7 30.25 3.36
0.7197 -0.49 Germ Weight (%) 17 11.00 1.00 7 11.45 1.56 0.4874
-0.45 Kernel Weight (g) 17 0.2141 0.0125 7 0.2114 0.0195 0.7365
0.0027 Kernel Oil (mg) 17 8.14 0.53 7 8.09 0.82 0.8807 0.05 Germ
Oil (mg) 17 6.25 0.80 7 6.56 1.24 0.5378 -0.31 ZM_S86572/LH244
Kernel Oil (%) 14 3.91 0.25 10 3.78 0.29 0.3110 0.14 Germ Weight
(g) 14 0.0220 0.0020 10 0.0212 0.0024 0.4270 0.0009 Germ Oil (%) 14
29.65 1.95 10 28.52 2.31 0.2892 1.12 Germ Weight (%) 14 12.21 0.97
10 11.33 1.15 0.1032 0.87 Kernel Weight (g) 14 0.2021 0.0095 10
0.2091 0.0112 0.1793 -0.0069 Kernel Oil (mg) 14 7.90 0.57 10 7.89
0.68 0.9605 0.02 Germ Oil (mg) 14 6.53 0.79 10 6.05 0.94 0.2713
0.47 ZM_S86578/LH244 Kernel Oil (%) 16 3.76 0.27 8 3.55 0.37 0.1921
0.21 Germ Weight (g) 16 0.0195 0.0013 8 0.0193 0.0019 0.7350 0.0003
Germ Oil (%) 16 29.78 1.51 8 27.82 2.14 0.0395 0.05 1.96 Germ
Weight (%) 16 11.66 0.81 8 10.89 1.15 0.1246 0.76 Kernel Weight (g)
16 0.1888 0.0104 8 0.1999 0.0146 0.0851 0.1 -0.0110 Kernel Oil (mg)
16 7.07 0.46 8 7.08 0.66 0.9640 -0.01 Germ Oil (mg) 16 5.80 0.36 8
5.34 0.51 0.0423 0.05 0.46 ZM_S87124/LH244 Kernel Oil (%) 13 2.55
0.29 11 3.57 0.31 <.0001 0.001 -1.03 Germ Weight (g) 13 0.0140
0.0011 11 0.0139 0.0012 0.8423 Germ Oil (%) 13 22.77 3.42 11 30.49
3.72 0.0002 0.001 -7.71 Germ Weight (%) 13 10.64 0.88 11 10.73 0.96
0.8354 -0.09 Kernel Weight (g) 13 0.1478 0.0100 11 0.1441 0.0108
0.4684 0.0037 Kernel Oil (mg) 13 3.76 0.48 11 5.15 0.52 <.0001
0.001 -1.39 Germ Oil (mg) 13 3.16 0.45 11 4.22 0.49 0.0001 0.001
-1.07
[0262] Examples of increased oil and organ size are demonstrated in
several events. The milligrams oil per germ was increased in two
independent events, ZM_S86578 (p=0.05) and ZM_S86487 (p=0.1). The
milligrams of oil per kernel (p=0.05) and kernel oil as a
percentage of dry wt (p=0.01) were both increased in event
ZM_S86481. Germ oil as a percentage of dry wt was increased in
three independent events, ZM_S86521 (p=0.1), ZM_S86548 (p=0.01) and
ZM_S86578 (p=0.05). Germ wt (gram dry wt) (p=0.1) and germ wt as a
percentage of total kernel wt (p=0.01) were increased in event
ZM_S86487.
[0263] Results from events containing pMON71279 are shown in Table
5.
TABLE-US-00005 TABLE 5 pMON71279 1st Generation Single Kernel
Analysis Positive GOI Negative GOI Variable n Mean LSD n Mean LSD
Prob > F Signif Delta LH244/ZM_S106956 Kernel Oil (%) 20 3.90
0.13 4 3.75 0.28 0.1840 0.15 Germ Weight (g) 20 0.0222 0.0019 4
0.0235 0.0043 0.4242 -0.0013 Germ Oil (%) 20 27.80 1.13 4 27.90
2.52 0.9210 -0.09 Germ Weight (%) 20 11.76 0.51 4 11.25 1.14 0.2463
0.51 Kernel Weight (g) 20 0.1885 0.0142 4 0.2078 0.0318 0.1197
-0.0193 Germ Oil (mg) 20 6.14 0.48 4 6.49 1.08 0.3952 -0.35 Kernel
Oil (mg) 20 7.32 0.51 4 7.78 1.14 0.2972 -0.45 LH244/ZM_S106984
Kernel Oil (%) 14 3.91 0.17 8 3.99 0.22 0.4023 -0.08 Germ Weight
(g) 14 0.0230 0.0021 8 0.0226 0.0028 0.7897 0.0003 Germ Oil (%) 14
28.84 1.57 8 29.29 2.08 0.6159 -0.45 Germ Weight (%) 14 11.66 0.90
8 11.31 1.19 0.4917 0.36 Kernel Weight (g) 14 0.1969 0.0114 8
0.2006 0.0151 0.5755 -0.0037 Germ Oil (mg) 14 6.61 0.59 8 6.61 0.79
0.9996 0.00 Kernel Oil (mg) 14 7.72 0.53 8 7.99 0.71 0.3879 -0.26
LH244/ZM_S106985 Kernel Oil (%) 12 3.56 0.18 11 3.38 0.19 0.0619
0.1 0.18 Germ Weight (g) 12 0.0211 0.0025 11 0.0214 0.0026 0.7901
-0.0003 Germ Oil (%) 12 25.42 1.90 11 25.14 1.98 0.7679 0.28 Germ
Weight (%) 12 10.80 1.06 11 11.11 1.11 0.5677 -0.30 Kernel Weight
(g) 12 0.1959 0.0107 11 0.1924 0.0111 0.5158 0.0035 Germ Oil (mg)
12 5.36 0.58 11 5.35 0.60 0.9574 0.02 Kernel Oil (mg) 12 6.98 0.53
11 6.48 0.55 0.0682 0.1 0.50 LH244/ZM_S108558 Kernel Oil (%) 14
3.60 0.17 10 3.73 0.20 0.1630 -0.13 Germ Weight (g) 14 0.0229
0.0012 10 0.0229 0.0015 0.9983 0.0000 Germ Oil (%) 14 26.20 1.48 10
26.71 1.75 0.5464 -0.52 Germ Weight (%) 14 11.90 0.66 10 11.85 0.78
0.8835 0.05 Kernel Weight (g) 14 0.1930 0.0137 10 0.1946 0.0163
0.8276 -0.0016 Germ Oil (mg) 14 6.02 0.47 10 6.11 0.56 0.7028 -0.10
Kernel Oil (mg) 14 6.92 0.48 10 7.28 0.56 0.1623 -0.36
ZM_S105162/LH244 Kernel Oil (%) 13 3.68 0.38 11 3.74 0.42 0.7919
-0.05 Germ Weight (g) 13 0.0193 0.0032 11 0.0179 0.0035 0.4090
0.0014 Germ Oil (%) 13 28.77 2.42 11 28.64 2.63 0.9174 0.13 Germ
Weight (%) 13 11.30 1.16 11 10.50 1.26 0.1872 0.80 Kernel Weight
(g) 13 0.1706 0.0226 11 0.1709 0.0246 0.9820 -0.0003 Germ Oil (mg)
13 5.50 0.83 11 5.12 0.90 0.3753 0.38 Kernel Oil (mg) 13 6.22 0.84
11 6.33 0.92 0.796 -0.11 ZM_S105166/LH244 Kernel Oil (%) 9 3.37
0.19 15 3.40 0.15 0.6861 -0.03 Germ Weight (g) 9 0.0216 0.0033 15
0.0182 0.0026 0.0288 0.05 0.0034 Germ Oil (%) 9 26.59 2.49 15 28.04
1.93 0.1921 -1.45 Germ Weight (%) 9 11.17 1.03 15 10.48 0.80 0.1391
0.68 Kernel Weight (g) 9 0.1933 0.0205 15 0.1721 0.0159 0.0255 0.05
0.0212 Germ Oil (mg) 9 5.71 0.92 15 5.09 0.71 0.1359 0.61 Kernel
Oil (mg) 9 6.49 0.79 15 5.88 0.61 0.0924 0.1 0.60 ZM_S105183/LH244
Kernel Oil (%) 18 3.30 0.19 6 3.28 0.33 0.8978 0.02 Germ Weight (g)
18 0.0214 0.0011 6 0.0192 0.0018 0.0059 0.01 0.0022 Germ Oil (%) 18
25.30 1.53 6 23.99 2.64 0.2221 1.31 Germ Weight (%) 18 11.65 0.58 6
10.95 1.00 0.0886 0.1 0.70 Kernel Weight (g) 18 0.1842 0.0086 6
0.1767 0.0149 0.2123 0.0075 Germ Oil (mg) 18 5.41 0.33 6 4.61 0.57
0.0017 0.01 0.80 Kernel Oil (mg) 18 6.06 0.39 6 5.77 0.67 0.2789
0.29 ZM_S105185/LH244 Kernel Oil (%) 19 3.36 0.15 5 3.22 0.29
0.2233 0.14 Germ Weight (g) 19 0.0201 0.0018 5 0.0188 0.0034 0.3493
0.0013 Germ Oil (%) 19 25.77 1.81 5 24.79 3.52 0.4769 0.98 Germ
Weight (%) 19 11.55 0.80 5 10.97 1.56 0.3447 0.58 Kernel Weight (g)
19 0.1743 0.0097 5 0.1714 0.0188 0.6905 0.0029 Germ Oil (mg) 19
5.12 0.28 5 4.67 0.55 0.0468 0.05 0.45 Kernel Oil (mg) 19 5.84 0.31
5 5.54 0.60 0.2058 0.30 ZM_S105187/LH244 Kernel Oil (%) 12 3.38
0.16 12 3.46 0.16 0.3052 -0.08 Germ Weight (g) 12 0.0196 0.0032 12
0.0192 0.0032 0.8001 0.0004 Germ Oil (%) 12 26.05 2.06 12 26.58
2.06 0.6010 -0.53 Germ Weight (%) 12 11.47 1.32 12 10.91 1.32
0.3872 0.56 Kernel Weight (g) 12 0.1695 0.0134 12 0.1748 0.0134
0.4212 -0.0053 Germ Oil (mg) 12 5.03 0.66 12 5.06 0.66 0.9253 -0.03
Kernel Oil (mg) 12 5.73 0.56 12 6.03 0.56 0.2684 -0.31
ZM_S105198/LH244 Kernel Oil (%) 3 3.27 0.37 21 3.34 0.14 0.6027
-0.07 Germ Weight (g) 3 0.0200 0.0027 21 0.0199 0.0010 0.9150
0.0001 Germ Oil (%) 3 24.09 3.68 21 25.97 1.39 0.1744 -1.88 Germ
Weight (%) 3 11.25 1.07 21 10.79 0.40 0.2510 0.46 Kernel Weight (g)
3 0.1783 0.0210 21 0.1845 0.0079 0.4210 -0.0063 Germ Oil (mg) 3
4.81 1.00 21 5.17 0.37 0.3244 -0.36 Kernel Oil (mg) 3 5.79 1.14 21
6.18 0.43 0.3587 -0.39 ZM_S106940/LH244 Kernel Oil (%) 18 3.77 0.17
5 3.62 0.32 0.2436 0.15 Germ Weight (g) 18 0.0174 0.0025 5 0.0150
0.0047 0.1983 0.0024 Germ Oil (%) 18 29.59 1.32 5 29.78 2.50 0.8476
-0.19 Germ Weight (%) 18 11.07 0.87 5 9.88 1.65 0.0752 0.1 1.19
Kernel Weight (g) 18 0.1563 0.0200 5 0.1544 0.0380 0.8979 0.0019
Germ Oil (mg) 18 5.13 0.73 5 4.46 1.38 0.2195 0.67 Kernel Oil (mg)
18 5.88 0.74 5 5.58 1.41 0.5874 0.30 ZM_S106960/LH244 Kernel Oil
(%) 12 3.88 0.20 12 3.78 0.20 0.3505 0.09 Germ Weight (g) 12 0.0218
0.0019 12 0.0215 0.0019 0.7616 0.0003 Germ Oil (%) 12 29.11 2.20 12
28.64 2.20 0.6596 0.47 Germ Weight (%) 12 11.32 0.82 12 10.86 0.82
0.2613 0.46 Kernel Weight (g) 12 0.1925 0.0091 12 0.1982 0.0091
0.2080 -0.0057 Germ Oil (mg) 12 6.32 0.58 12 6.15 0.58 0.5547 0.17
Kernel Oil (mg) 12 7.48 0.45 12 7.49 0.45 0.9759 -0.01
ZM_S106965/LH244 Kernel Oil (%) 14 3.86 0.17 10 3.72 0.21 0.1510
0.14 Germ Weight (g) 14 0.0217 0.0020 10 0.0219 0.0024 0.8803
-0.0002 Germ Oil (%) 14 29.87 1.65 10 29.17 1.95 0.4270 0.71 Germ
Weight (%) 14 11.39 0.69 10 11.22 0.81 0.6391 0.17 Kernel Weight
(g) 14 0.1912 0.0157 10 0.1948 0.0186 0.6752 -0.0035 Germ Oil (mg)
14 6.49 0.63 10 6.36 0.74 0.7067 0.13 Kernel Oil (mg) 14 7.39 0.75
10 7.24 0.88 0.7115 0.15 ZM_S106971/LH244 Kernel Oil (%) 12 3.73
0.25 11 3.88 0.26 0.2432 -0.15 Germ Weight (g) 12 0.0219 0.0017 11
0.0229 0.0018 0.2563 -0.0010 Germ Oil (%) 12 29.02 1.49 11 28.30
1.56 0.3377 0.72 Germ Weight (%) 12 11.00 0.78 11 11.37 0.81 0.3351
-0.38 Kernel Weight (g) 12 0.1998 0.0133 11 0.2020 0.0139 0.7431
-0.0022 Germ Oil (mg) 12 6.36 0.57 11 6.48 0.59 0.6736 -0.12 Kernel
Oil (mg) 12 7.48 0.80 11 7.88 0.83 0.3201 -0.40 ZM_S106973/LH244
Kernel Oil (%) 8 4.11 0.46 16 4.16 0.33 0.8231 -0.04 Germ Weight
(g) 8 0.0169 0.0055 16 0.0170 0.0039 0.972 -0.0001 Germ Oil (%) 8
28.48 3.52 16 30.06 2.49 0.2951 -1.57 Germ Weight (%) 8 11.42 1.59
16 10.92 1.12 0.4593 0.50 Kernel Weight (g) 8 0.1448 0.0397 16
0.1532 0.0281 0.6167 -0.0084 Germ Oil (mg) 8 4.88 1.82 16 5.13 1.29
0.746 -0.25 Kernel Oil (mg) 8 6.03 1.94 16 6.40 1.37 0.6506 -0.37
ZM_S106979/LH244 Kernel Oil (%) 7 3.99 0.23 17 3.98 0.15 0.9716
0.00 Germ Weight (g) 7 0.0244 0.0044 17 0.0190 0.0028 0.006 0.01
0.0054 Germ Oil (%) 7 30.95 1.90 17 31.51 1.22 0.4717 -0.56 Germ
Weight (%) 7 11.49 1.09 17 11.19 0.70 0.511 0.30 Kernel Weight (g)
7 0.2139 0.0382 17 0.1702 0.0245 0.0098 0.01 0.0437 Germ Oil (mg) 7
7.52 1.32 17 5.98 0.85 0.0088 0.01 1.54 Kernel Oil (mg) 7 8.53 1.60
17 6.78 1.02 0.013 0.05 1.75 ZM_S106982/LH244 Kernel Oil (%) 13
3.86 0.21 11 3.96 0.23 0.3465 -0.10 Germ Weight (g) 13 0.0217
0.0025 11 0.0210 0.0027 0.5821 0.0007 Germ Oil (%) 13 28.08 1.66 11
29.70 1.80 0.0644 0.1 -1.62 Germ Weight (%) 13 11.75 0.93 11 11.41
1.01 0.4732 0.34 Kernel Weight (g) 13 0.1841 0.0170 11 0.1849
0.0185 0.9244 -0.0008 Germ Oil (mg) 13 6.09 0.76 11 6.22 0.83
0.7215 -0.14 Kernel Oil (mg) 13 7.08 0.61 11 7.31 0.67 0.4652 -0.23
ZM_S106983/LH244 Kernel Oil (%) 14 3.97 0.15 8 4.03 0.20 0.5323
-0.05 Germ Weight (g) 14 0.0225 0.0020 8 0.0212 0.0026 0.2314
0.0014 Germ Oil (%) 14 30.32 1.12 8 30.54 1.49 0.7329 -0.22 Germ
Weight (%) 14 11.77 0.61 8 11.93 0.80 0.6405 -0.16 Kernel Weight
(g) 14 0.1911 0.0140 8 0.1780 0.0185 0.1093 0.1 0.0132 Germ Oil
(mg) 14 6.83 0.64 8 6.47 0.85 0.3314 0.36 Kernel Oil (mg) 14 7.61
0.61 8 7.16 0.81 0.2031 0.45
[0264] Examples of increased oil and organ size are demonstrated in
several events. Germ wt (g dry wt) was increased in three
independent events, ZM_S105183 (p=0.01); ZM_S105166 (p=0.05) and
ZM_S106973 (p=0.01). The milligrams of oil per germ was increased
in three independent events, ZM_S105183 (p=0.01), ZM_S105185
(p=0.05), and ZM_S106979 (p=0.01). Germ wt as a percentage of total
kernel wt was increased in two independent events, ZM_S105183
(p=0.1) and ZM_S106940 (p=0.1). The milligrams of oil per kernel
was increased in two independent events, ZM_S105166 (p0.1) and
ZM_S106979 (p=0.05). Kernel oil as a percentage of dry wt was
increased in event ZM_S106985 (p=0.1). Kernel wt (g dry wt) was
increased in two independent events ZM_S105166 (p=0.05) and
ZM_S106979 (p=0.01).
[0265] Results show that seed harboring the pMON71270, and other
constructs described in Examples 3 and 4, have at least one of the
following phenotypes: increased germ mass (as a percent of kernel
weight), increased whole kernel oil levels (as a percent of kernel
weight), improved protein quality (increased lysine, tryptophan,
threonine, isoleucine and methionine, for example), free amino
acids (up or down) or increased micronutrient content, including
tocopherols, tocotrienols, carotenoids, plastiquinols and
phylloquinones.
Example 13
[0266] This example describes the histological analysis of tissues
from transgenic maize plants.
[0267] Mature or developing kernels of fresh or frozen corn are
sliced sagitally. Tissue slices of 1 mm or thinner are stained for
1 to 2 min in a solution of 0.01% aniline blue C.I. 42755 (Baker,
Phillipsburg, N.J.) in 0.1M phosphate buffer, pH 8.0. Samples are
rinsed in 0.1 M phosphate buffer, pH 8.0, and examined with either
of a standard fluorescent microscope using a narrow violet cube
Nikon V-4 (EX 380-420 nm, DM 430 nm, BA 450 nm) or an ultraviolet
light source (EX330-380, DM400, BA420). Alternatively, samples can
be visualized using a confocal microscope using an Argon-ion laser
(emission wavelength 488 nm and 514 nm). Aniline blue stains the
cell walls and nuclei of plants (Smith and McCully, Stain
Technology, 53(2):79-85 (1978)). Cell size is determined by using
an outlining tool in the Nikon EZ C-1 software. Approximately 25 to
50 cells are outlined and the area the cells occupy is recorded and
the average area per cell is calculated.
[0268] Histological analysis of events containing pMON71270 yielded
3 events that demonstrated a significant increase in cell number as
characterized by more cells per unit area in the embryo
(1.3.times., 1.7.times., 2.4.times., p=0.05) with no reduction in
embryo mass.
[0269] Histological analysis of events containing pMON71279 gave 3
events with wild-type cell number per unit area (1.1.times.,
0.97.times., 0.96.times., p=0.05), but due to increased embryo
mass, yielded an increase in the total number of cells per
embryo.
[0270] To determine mitotic indices, corn tissues are fixed,
stained with a fluorescent probe, cleared and imaged using
fluorescent microscopy or confocal scanning laser microscopy
(CLSM). Mitotic index is determined by counting the number of cells
in an area that are in prometaphase, metaphase or anaphase and
reported as a percentage. For example, corn tissue can be fixed in
formalin, propinionic acid and ethanol, stained with propidium
iodide and cleared with xylene (Running et al., Confocal Microscopy
of the Shoot Apex, Methods in Cell Biology, 49:217-229 (1995)).
Example 14
[0271] This example describes how to measure CDK inhibitor
activity.
[0272] Protein extracts from transgenic and non-transgenic plant
tissues are enriched for CDK using p13Suc1 resin (Oncogene
Sciences, Cambridge, Mass.), and histone H1 kinase assays are
performed (Wang et al., Plant J., 15:501-510 (1998)). Transgenic
plant tissues with increased CDK inhibitor activity have reduced
kinase activity, whereas transgenic plant tissues with reduced CDK
inhibitor activity have increased kinase activity.
[0273] The following references are specifically incorporated
herein by reference: [0274] Birren et al., Genome Analysis: A
Laboratory Manual Series, Volume 1, Analyzing DNA, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1997), [0275]
Birren et al., Genome Analysis: A Laboratory Manual Series, Volume
2, Detecting Genes, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1998), [0276] Birren et al., Genome Analysis:
A Laboratory Manual Series, Volume 3, Cloning Systems, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), [0277]
Birren et al., Genome Analysis: A Laboratory Manual Series, Volume
4, Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1999), [0278] Harlow et al., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1988), [0279] Harlow et al., Using Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1999), [0280] Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989), [0281] Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley & Sons,
Inc., NY (1999), and [0282] Croy, Plant Molecular Biology Labfase,
BIOS Scientific Publications, Ltd. (UK) and Blackwell Scientific
Publications (UK) (1993).
[0283] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
While this invention has been described with an emphasis upon
preferred embodiments, it will be apparent to those of ordinary
skill in the art that variations in the preferred embodiments can
be prepared and used and that the invention can be practiced
otherwise than as specifically described herein. The present
invention is intended to include such variations and alternative
practices. Accordingly, this invention includes all modifications
encompassed within the spirit and scope of the invention as defined
by the following claims.
[0284] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0285] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
591642DNAZea mays 1atggggaagt acatgcgcaa gggcaaggtg tccggggagg
tcgccgtcat ggaggtaccc 60ggcggcgcgc tgctcggcgt ccgcacccgc tcccgcacgc
tcgcgctgca gcgcgcgcag 120aggccgctcg acaagggcga cgcggaggac
gccgccgcgg agtacctcga gctcaggagc 180cggaggctcg agaagccgca
caaggagcat ccgtcgccgc ccgcgaccgc gaccaagagg 240ggcgccggga
ggaaggccgc cgccgccgcc gcggtgcagc acgtgctgat gcaggacgag
300gtcgaggtcg aggtctcgtt cggggacaac gtgcttgact tggacaccat
ggaaaggagt 360accagagaga caacaccgtg cagcctgatt aggaacccag
agatgataag caccccagga 420tccacaacta aaagcaaaac cagcagcaac
tcgacgactt cccgccgcag aacggaggaa 480accccgagct gccggttcat
accgagctcg ctcgagatgg aggagttctt ctcggcggcc 540gagcaacagg
agcagcatag cttcagggag aagtacaact tctgtcccgt gaacgactgt
600cctctccctg gccggtacga atgggcgagg ctagactgct ag 6422213PRTZea
mays 2Met Gly Lys Tyr Met Arg Lys Gly Lys Val Ser Gly Glu Val Ala
Val1 5 10 15Met Glu Val Pro Gly Gly Ala Leu Leu Gly Val Arg Thr Arg
Ser Arg 20 25 30Thr Leu Ala Leu Gln Arg Ala Gln Arg Pro Leu Asp Lys
Gly Asp Ala 35 40 45Glu Asp Ala Ala Ala Glu Tyr Leu Glu Leu Arg Ser
Arg Arg Leu Glu 50 55 60Lys Pro His Lys Glu His Pro Ser Pro Pro Ala
Thr Ala Thr Lys Arg65 70 75 80Gly Ala Gly Arg Lys Ala Ala Ala Ala
Ala Ala Val Gln His Val Leu 85 90 95Met Gln Asp Glu Val Glu Val Glu
Val Ser Phe Gly Asp Asn Val Leu 100 105 110Asp Leu Asp Thr Met Glu
Arg Ser Thr Arg Glu Thr Thr Pro Cys Ser 115 120 125Leu Ile Arg Asn
Pro Glu Met Ile Ser Thr Pro Gly Ser Thr Thr Lys 130 135 140Ser Lys
Thr Ser Ser Asn Ser Thr Thr Ser Arg Arg Arg Thr Glu Glu145 150 155
160Thr Pro Ser Cys Arg Phe Ile Pro Ser Ser Leu Glu Met Glu Glu Phe
165 170 175Phe Ser Ala Ala Glu Gln Gln Glu Gln His Ser Phe Arg Glu
Lys Tyr 180 185 190Asn Phe Cys Pro Val Asn Asp Cys Pro Leu Pro Gly
Arg Tyr Glu Trp 195 200 205Ala Arg Leu Asp Cys 2103654DNAZea mays
3atggggaagt acatgcgcaa gggcaagatg tccggggagg tggccgtcat ggaggtcccc
60ggcggcgcgc tgctgggtgt ccgcacccgc tcccgcacgc tcgcgctgca gagggcgcag
120aggccgctcg acaaggggga cgcggatgac gccgccggac agtacctcga
gctcaggagc 180cggaggctcg agaagcctca taaggaccat cagccgctgc
cgctgccgct gccgccgccc 240gcccccgcag ccaagagggg cgccgggagg
aaggccgcct ccaccgccgc cgcgccaaac 300gcgctggcgg aggacgaggt
cgaggtcgag gtctccttcg gggagaacgt gcttgacttg 360gacgccatgg
aaaggagtac cagagagaca acaccgtgta gtttgatcag gaacccagag
420atgataagca ccccaggatc cacaactaaa agtaaaacca gcaactcgac
gacttcccgt 480cgcaggatgg aaacctcagt ctgccgtttc ataccgagtt
cgctcgagat ggaagagttc 540ttctcggccg ctgaacaaca ggagcagcat
aacttcaggg agaagtataa cttctgtcct 600gtgaacgact gccctctccc
gggtcgatat gagtgggcga ggctagactg ttag 6544217PRTZea mays 4Met Gly
Lys Tyr Met Arg Lys Gly Lys Met Ser Gly Glu Val Ala Val1 5 10 15Met
Glu Val Pro Gly Gly Ala Leu Leu Gly Val Arg Thr Arg Ser Arg 20 25
30Thr Leu Ala Leu Gln Arg Ala Gln Arg Pro Leu Asp Lys Gly Asp Ala
35 40 45Asp Asp Ala Ala Gly Gln Tyr Leu Glu Leu Arg Ser Arg Arg Leu
Glu 50 55 60Lys Pro His Lys Asp His Gln Pro Leu Pro Leu Pro Leu Pro
Pro Pro65 70 75 80Ala Pro Ala Ala Lys Arg Gly Ala Gly Arg Lys Ala
Ala Ser Thr Ala 85 90 95Ala Ala Pro Asn Ala Leu Ala Glu Asp Glu Val
Glu Val Glu Val Ser 100 105 110Phe Gly Glu Asn Val Leu Asp Leu Asp
Ala Met Glu Arg Ser Thr Arg 115 120 125Glu Thr Thr Pro Cys Ser Leu
Ile Arg Asn Pro Glu Met Ile Ser Thr 130 135 140Pro Gly Ser Thr Thr
Lys Ser Lys Thr Ser Asn Ser Thr Thr Ser Arg145 150 155 160Arg Arg
Met Glu Thr Ser Val Cys Arg Phe Ile Pro Ser Ser Leu Glu 165 170
175Met Glu Glu Phe Phe Ser Ala Ala Glu Gln Gln Glu Gln His Asn Phe
180 185 190Arg Glu Lys Tyr Asn Phe Cys Pro Val Asn Asp Cys Pro Leu
Pro Gly 195 200 205Arg Tyr Glu Trp Ala Arg Leu Asp Cys 210
2155342DNAZea mays 5ttatcagtca tcacctccca actcgcaacg cacccacagt
ccctcaccgg ctcaccgccc 60gcaccgcaca caggcgccac gaagcagaaa agtatggccg
ctgccacagc gacggcggcg 120gtgctgggat gcagcaggcg ccagagcgac
attgcgggcg ccggcatacc gaagaaggga 180aaggtgggga ggtcgccgcc
ggcggaggag gtggaggcgt tcctcgccgc agcggagcgc 240ggcatggcgc
ggcgcttcgc ggtcaagtac aactatgacg tcgtcaagga cgctcccatg
300gacggcggcc ggtacgagtg ggtccgagtg cggcccggtt aa 3426129PRTZea
mays 6Ala Cys Arg Tyr Arg Ser Gly Ile Pro Gly Ser Thr His Ala Ser
Ala1 5 10 15Leu Ser Val Ile Thr Ser Gln Leu Ala Thr His Pro Gln Ser
Leu Thr 20 25 30Gly Ser Pro Pro Ala Pro His Thr Gly Ala Thr Lys Gln
Lys Ser Met 35 40 45Ala Ala Ala Thr Ala Thr Ala Ala Val Leu Gly Cys
Ser Arg Arg Gln 50 55 60Ser Asp Ile Ala Gly Ala Gly Ile Pro Lys Lys
Gly Lys Val Gly Arg65 70 75 80Ser Pro Pro Ala Glu Glu Val Glu Ala
Phe Leu Ala Ala Ala Glu Arg 85 90 95Gly Met Ala Arg Arg Phe Ala Val
Lys Tyr Asn Tyr Asp Val Val Lys 100 105 110Asp Ala Pro Met Asp Gly
Gly Arg Tyr Glu Trp Val Arg Val Arg Pro 115 120 125Gly7573DNAZea
mays 7atgggcaagt acatgcgcaa ggccaaggct tccagcgagg ttgtcatcat
ggatgtcgcc 60gccgctccgc tcggagtccg cacccgagcg cgcgccctcg cgctgcagcg
tctgcaggag 120caacagacgc agtgggaaga aggtgctggc ggcgagtacc
tggagctaag gaaccggagg 180ctcgagaagc tgccgccgcc gccggcgacc
actaggaggt cgggcgggag gaaagcggca 240gccgaggccg ccgcaactaa
ggaggctgag gcgtcgtacg gggagaacat gctcgagttg 300gaggccatgg
agaggattac cagggagacg acgccttgca gcttgattaa cacccagatg
360actagcactc ctgggtccac gagatccagc cactcttgcc accgcagggt
gaacgctcct 420ccggtgcacg ccgtcccaag ttctagggag atgaatgagt
acttcgctgc cgaacagcga 480cggcaacagc aggatttcat tgacaagtac
aacttcgatc ctgcaaacga ctgccctctc 540ccaggcaggt ttgagtgggt
gaagctagac tga 5738190PRTZea mays 8Met Gly Lys Tyr Met Arg Lys Ala
Lys Ala Ser Ser Glu Val Val Ile1 5 10 15Met Asp Val Ala Ala Ala Pro
Leu Gly Val Arg Thr Arg Ala Arg Ala 20 25 30Leu Ala Leu Gln Arg Leu
Gln Glu Gln Gln Thr Gln Trp Glu Glu Gly 35 40 45Ala Gly Gly Glu Tyr
Leu Glu Leu Arg Asn Arg Arg Leu Glu Lys Leu 50 55 60Pro Pro Pro Pro
Ala Thr Thr Arg Arg Ser Gly Gly Arg Lys Ala Ala65 70 75 80Ala Glu
Ala Ala Ala Thr Lys Glu Ala Glu Ala Ser Tyr Gly Glu Asn 85 90 95Met
Leu Glu Leu Glu Ala Met Glu Arg Ile Thr Arg Glu Thr Thr Pro 100 105
110Cys Ser Leu Ile Asn Thr Gln Met Thr Ser Thr Pro Gly Ser Thr Arg
115 120 125Ser Ser His Ser Cys His Arg Arg Val Asn Ala Pro Pro Val
His Ala 130 135 140Val Pro Ser Ser Arg Glu Met Asn Glu Tyr Phe Ala
Ala Glu Gln Arg145 150 155 160Arg Gln Gln Gln Asp Phe Ile Asp Lys
Tyr Asn Phe Asp Pro Ala Asn 165 170 175Asp Cys Pro Leu Pro Gly Arg
Phe Glu Trp Val Lys Leu Asp 180 185 1909651DNAZea mays 9atgggcaagt
gcgtgaggat ccgcggcagc agcaagccgc gcgccgccgc cgccgcggcg 60gcgtcgtgcc
tcacgctgtg cagcgggcgc cgcgtgccgc cgtcggaggc gtcggcggcg
120tgcagcccga ggacgagaag caggccgcgg cgccaccgcg gggcaggcct
ccggcggtgg 180tgcggcgcca aggagagcgc gtacggcggc agcccccggc
gccacagggg cgagggcgag 240gccgacgcgc ggagcccccg tggccgggtg
ctcggtgtcg gtgcccgcca gcagcagctc 300tgcgccgacg acgggctcgg
ccagcagcac gaggaggagg cgtctgcgac gatggccggc 360gactgcgacg
acggcgcggg cgtggcgaaa gtgaataagg cgaataaaca cgagaacgac
420gagtgcggct gccgcgtcgt cggcggcgtc gccagccaga cgccgtcgcc
gtcgccgtcg 480ccgccaccgc cgccgacgga aaccgagata gaggccttct
tcgcggacgc ggagctggcc 540gagcgccggc gattcgcaga ggcgtacaat
tacgacgtcg ccctcgaccg cccgctggag 600gggcgcttcg agtgggtgcc
gctgccgctg acggggggtc ggaggtggta a 65110216PRTZea mays 10Met Gly
Lys Cys Val Arg Ile Arg Gly Ser Ser Lys Pro Arg Ala Ala1 5 10 15Ala
Ala Ala Ala Ala Ser Cys Leu Thr Leu Cys Ser Gly Arg Arg Val 20 25
30Pro Pro Ser Glu Ala Ser Ala Ala Cys Ser Pro Arg Thr Arg Ser Arg
35 40 45Pro Arg Arg His Arg Gly Ala Gly Leu Arg Arg Trp Cys Gly Ala
Lys 50 55 60Glu Ser Ala Tyr Gly Gly Ser Pro Arg Arg His Arg Gly Glu
Gly Glu65 70 75 80Ala Asp Ala Arg Ser Pro Arg Gly Arg Val Leu Gly
Val Gly Ala Arg 85 90 95Gln Gln Gln Leu Cys Ala Asp Asp Gly Leu Gly
Gln Gln His Glu Glu 100 105 110Glu Ala Ser Ala Thr Met Ala Gly Asp
Cys Asp Asp Gly Ala Gly Val 115 120 125Ala Lys Val Asn Lys Ala Asn
Lys His Glu Asn Asp Glu Cys Gly Cys 130 135 140Arg Val Val Gly Gly
Val Ala Ser Gln Thr Pro Ser Pro Ser Pro Ser145 150 155 160Pro Pro
Pro Pro Pro Thr Glu Thr Glu Ile Glu Ala Phe Phe Ala Asp 165 170
175Ala Glu Leu Ala Glu Arg Arg Arg Phe Ala Glu Ala Tyr Asn Tyr Asp
180 185 190Val Ala Leu Asp Arg Pro Leu Glu Gly Arg Phe Glu Trp Val
Pro Leu 195 200 205Pro Leu Thr Gly Gly Arg Arg Trp 210
21511765DNAZea mays 11atggggaagt acatgcgcaa gtgcaggggc gccgcaggcg
cggaggtcgc cgccgtcgag 60gttacgcagg tcgtcggcgt ccggacgagg tccaggtccg
cggcggcgac cggcggtgtc 120gcgaaggtcg ccccgaggag gaagagggcg
ccggcggggg arcctgctgc cgccgtgagc 180gctggtgggg acggcggaag
ctgctacatc cacctgcgta gccgcatgct gttcatggca 240ccgcctcagc
cgcagccgtc ggttccgacc ccggcggcgg aggctgctga tggcgctgca
300ggacagcagg gcgcggtgct cgcggccggg ctctcgcgct gctccagcac
ggcgtcgtcg 360gtgaacttgg ggttgggggg tcagcgcggg agccacacct
gccgctccga cgacgctgca 420gaggctggcg gggatcacgt cctggtggtg
gatgtctcgg cgagcaactc cgggagcggc 480ccagaccgcg agaggagaga
gacgacgcca tcgagccggg cgcacggcga gctcagcgat 540ctggagtcgg
atctggcggg gcacaagact ggcccgtcgc taccggcggc aacgccggct
600gcggagctga tcgtgccgcc agcacacgag atccaggagt tcttcgccgc
cgccgaggcg 660gcccaggcca agcgctttgc ttccaagtac aacttcgact
tcgtccgcgg cgtgcccctc 720gacgccggcg gccggttcga gtgggcgccg
gtggtcagca tctga 76512254PRTZea mays 12Met Gly Lys Tyr Met Arg Lys
Cys Arg Gly Ala Ala Gly Ala Glu Val1 5 10 15Ala Ala Val Glu Val Thr
Gln Val Val Gly Val Arg Thr Arg Ser Arg 20 25 30Ser Ala Ala Ala Thr
Gly Gly Val Ala Lys Val Ala Pro Arg Arg Lys 35 40 45Arg Ala Pro Ala
Gly Glu Pro Ala Ala Ala Val Ser Ala Gly Gly Asp 50 55 60Gly Gly Ser
Cys Tyr Ile His Leu Arg Ser Arg Met Leu Phe Met Ala65 70 75 80Pro
Pro Gln Pro Gln Pro Ser Val Pro Thr Pro Ala Ala Glu Ala Ala 85 90
95Asp Gly Ala Ala Gly Gln Gln Gly Ala Val Leu Ala Ala Gly Leu Ser
100 105 110Arg Cys Ser Ser Thr Ala Ser Ser Val Asn Leu Gly Leu Gly
Gly Gln 115 120 125Arg Gly Ser His Thr Cys Arg Ser Asp Asp Ala Ala
Glu Ala Gly Gly 130 135 140Asp His Val Leu Val Val Asp Val Ser Ala
Ser Asn Ser Gly Ser Gly145 150 155 160Pro Asp Arg Glu Arg Arg Glu
Thr Thr Pro Ser Ser Arg Ala His Gly 165 170 175Glu Leu Ser Asp Leu
Glu Ser Asp Leu Ala Gly His Lys Thr Gly Pro 180 185 190Ser Leu Pro
Ala Ala Thr Pro Ala Ala Glu Leu Ile Val Pro Pro Ala 195 200 205His
Glu Ile Gln Glu Phe Phe Ala Ala Ala Glu Ala Ala Gln Ala Lys 210 215
220Arg Phe Ala Ser Lys Tyr Asn Phe Asp Phe Val Arg Gly Val Pro
Leu225 230 235 240Asp Ala Gly Gly Arg Phe Glu Trp Ala Pro Val Val
Ser Ile 245 25013717DNAZea mays 13gtcgccgccg tcgaggttac gcaggtcgtc
ggcgtccgca cgaggtccag gtccgcggcg 60gcgaccggcg gtgtcgcgaa ggtcgtcgcc
ccgaggagga agagggcgcc ggcgggggag 120cctgctgcct ccgtgggcgc
tggtggggac ggcggaagct gctacatcca cctgcgtagc 180cgcatgctgt
tcatggcacc gcctcagccg cagccgccgt cggttccgac cccggcggag
240gctgctgatg gcgctgcagg acagcagggc gcggcgctcg cggccgggct
ctcgcgttgc 300tccagcacgg cgtcgtcggt gcacgtgggg ggtcagcgcg
ggagccacac ctgccgctcc 360gacgacgctg cagaggctgg cggggatcac
gtcctggtgg atgtctcggc ggcgagcaac 420tccgggagcg gcccagaccg
cgagaggcga gagacgacgc catcgagccg ggcgcacggc 480gagctcagcg
atctggagtc ggatctggcg gggcacaaga ctggcccgtc gctaccggcg
540gcaacgccgg ctgcggagct gatcgtgccg ccagcacacg agatccagga
gttcttcgcc 600gccgccgagg cggcccaggc caagcgcttt gcttccaagt
acaacttcga cttcgtccgt 660ggcgtgcccc tcgacgccgg cggccggttc
gagtgggcgc cggtggtcag catctga 71714238PRTZea mays 14Val Ala Ala Val
Glu Val Thr Gln Val Val Gly Val Arg Thr Arg Ser1 5 10 15Arg Ser Ala
Ala Ala Thr Gly Gly Val Ala Lys Val Val Ala Pro Arg 20 25 30Arg Lys
Arg Ala Pro Ala Gly Glu Pro Ala Ala Ser Val Gly Ala Gly 35 40 45Gly
Asp Gly Gly Ser Cys Tyr Ile His Leu Arg Ser Arg Met Leu Phe 50 55
60Met Ala Pro Pro Gln Pro Gln Pro Pro Ser Val Pro Thr Pro Ala Glu65
70 75 80Ala Ala Asp Gly Ala Ala Gly Gln Gln Gly Ala Ala Leu Ala Ala
Gly 85 90 95Leu Ser Arg Cys Ser Ser Thr Ala Ser Ser Val His Val Gly
Gly Gln 100 105 110Arg Gly Ser His Thr Cys Arg Ser Asp Asp Ala Ala
Glu Ala Gly Gly 115 120 125Asp His Val Leu Val Asp Val Ser Ala Ala
Ser Asn Ser Gly Ser Gly 130 135 140Pro Asp Arg Glu Arg Arg Glu Thr
Thr Pro Ser Ser Arg Ala His Gly145 150 155 160Glu Leu Ser Asp Leu
Glu Ser Asp Leu Ala Gly His Lys Thr Gly Pro 165 170 175Ser Leu Pro
Ala Ala Thr Pro Ala Ala Glu Leu Ile Val Pro Pro Ala 180 185 190His
Glu Ile Gln Glu Phe Phe Ala Ala Ala Glu Ala Ala Gln Ala Lys 195 200
205Arg Phe Ala Ser Lys Tyr Asn Phe Asp Phe Val Arg Gly Val Pro Leu
210 215 220Asp Ala Gly Gly Arg Phe Glu Trp Ala Pro Val Val Ser
Ile225 230 23515699DNAZea mays 15atggggaagt acatgcgcaa gcgcaggggg
gccgcgggcg agggggtggc cgcagtcgag 60gtctcgcagg tcgtcggcgt ccggacgagg
tccaggtccg cggcggcgac cggcggcggt 120gtcgcgaagg tcgctccgcc
gaggaggaag aaggcgctgc tgcccgccgc gaacgagacg 180gcgtcggggg
agcctggtgc cgtgggcggt ggtggtgggg acggcggaag ctgctgctac
240atccacctgc ggagccgcat gctgttcatg gcagcacctc agcagcaacc
gtcggcggct 300ccgacgcccg cggaggctgc tggtgcggca cagcagggcg
gggtggtggc gctcgcggct 360ggcctctcgc gttgctccag cacggcgtcg
acggtggacg tcgggggcca gcagcccgcg 420agcgggagcc acgcctgccg
ctccgacgct gcggaggttg ccggggatca cgtcccggat 480gtcgtcaccg
cgagcaactc ggggagcgtc ccggaccgcg agaggagaga gacgacgcca
540tcgtcgagcc gggcgcacgg cggcgagctc agcgatctgg agtcggatct
ggtggggtgg 600cagaagactg gctgctcgtc gtcgccggcg acaacaacgt
cggctgcgga gctgatcgtg 660ccgccagcac aggagatcca ggaattcttc gcggccgct
69916232PRTZea mays 16Met Gly Lys Tyr Met Arg Lys Arg Arg Gly Ala
Ala Gly Glu Gly Val1 5 10 15Ala Ala Val Glu Val Ser Gln Val Val Gly
Val Arg Thr Arg Ser Arg 20 25 30Ser Ala Ala Ala Thr Gly Gly Gly Val
Ala Lys Val Ala Pro Pro Arg 35 40 45Arg Lys Lys Ala Leu Leu Pro Ala
Ala Asn Glu Thr Ala Ser Gly Glu 50 55 60Pro Gly Ala Val Gly Gly Gly
Gly Gly Asp Gly Gly Ser Cys Cys Tyr65 70 75 80Ile His Leu Arg Ser
Arg Met Leu Phe Met Ala Ala Pro Gln Gln Gln 85 90 95Pro Ser Ala Ala
Pro Thr Pro Ala Glu Ala Ala Gly Ala Ala Gln Gln 100 105 110Gly Gly
Val Val Ala Leu Ala Ala Gly Leu Ser Arg Cys Ser Ser Thr 115 120
125Ala Ser Thr Val Asp Val Gly Gly Gln Gln Pro Ala Ser Gly Ser His
130 135 140Ala Cys Arg Ser Asp Ala
Ala Glu Val Ala Gly Asp His Val Pro Asp145 150 155 160Val Val Thr
Ala Ser Asn Ser Gly Ser Val Pro Asp Arg Glu Arg Arg 165 170 175Glu
Thr Thr Pro Ser Ser Ser Arg Ala His Gly Gly Glu Leu Ser Asp 180 185
190Leu Glu Ser Asp Leu Val Gly Trp Gln Lys Thr Gly Cys Ser Ser Ser
195 200 205Pro Ala Thr Thr Thr Ser Ala Ala Glu Leu Ile Val Pro Pro
Ala Gln 210 215 220Glu Ile Gln Glu Phe Phe Ala Ala225
23017183DNAZea mays 17cactcttccc accgcagggt gaaagctcct cctgtgcacg
ccctcccaag ttcaacggag 60atgaacgagt acttcgctgc tgaacagcga cgccaacaac
aggctttcat tgacaagtac 120aactttgatc ctgtaaatga ctgccctctc
ccaggcaggt ttgaatgggt gaagctagac 180tga 1831860PRTZea mays 18His
Ser Ser His Arg Arg Val Lys Ala Pro Pro Val His Ala Leu Pro1 5 10
15Ser Ser Thr Glu Met Asn Glu Tyr Phe Ala Ala Glu Gln Arg Arg Gln
20 25 30Gln Gln Ala Phe Ile Asp Lys Tyr Asn Phe Asp Pro Val Asn Asp
Cys 35 40 45Pro Leu Pro Gly Arg Phe Glu Trp Val Lys Leu Asp 50 55
6019200DNAArtificialPrimer 19gcagcaactc gacgacttcc cgccgcagaa
cggaggaaac cccgagctgc cggttcatac 60cgagctcgct cgagatggag gagttcttct
cggcggccga gcaacaggag cagcatagct 120tcagggagaa gtacaacttc
tgtcccgtga acgactgtcc tctccctggc cggtacgaat 180gggcgaggct
agactgctag 20020200DNAArtificialPrimer 20aactaaaagt aaaaccagca
actcgacgac ttcccgtcgc aggatggaaa cctcagtctg 60ccgtttcata ccgagttcgc
tcgagatgga agagttcttc tcggccgctg aacaacagga 120gcagcataac
ttcagggaga agtataactt ctgtcctgtg aacgactgcc ctctcccggg
180tcgatatgag tgggcgaggc 20021200DNAArtificialPrimer 21agagcgacat
tgcgggcgcc ggcataccga agaagggaaa ggtggggagg tcgccgccgg 60cggaggaggt
ggaggcgttc ctcgccgcag cggagcgcgg catggcgcgg cgcttcgcgg
120tcaagtacaa ctatgacgtc gtcaaggacg ctcccatgga cggcggccgg
tacgagtggg 180tccgagtgcg gcccggttaa 20022200DNAArtificialPrimer
22ggtccacgag atccagccac tcttgccacc gcagggtgaa cgctcctccg gtgcacgccg
60tcccaagttc tagggagatg aatgagtact tcgctgccga acagcgacgg caacagcagg
120atttcattga caagtacaac ttcgatcctg caaacgactg ccctctccca
ggcaggtttg 180agtgggtgaa gctagactga 20023200DNAArtificialPrimer
23ccagccagac gccgtcgccg tcgccgtcgc cgccaccgcc gccgacggaa accgagatag
60aggccttctt cgcggacgcg gagctggccg agcgccggcg attcgcagag gcgtacaatt
120acgacgtcgc cctcgaccgc ccgctggagg ggcgcttcga gtgggtgccg
ctgccgctga 180cggggggtcg gaggtggtaa 20024200DNAArtificialPrimer
24ccatcgagcc gggcgcacgg cgagctcagc gatctggagt cggatctggc ggggcacaag
60actggcccgt cgctaccggc ggcaacgccg gctgcggagc tgatcgtgcc gccagcacac
120gagatccagg agttcttcgc cgccgccgag gcggcccagg ccaagcgctt
tgcttccaag 180tacaacttcg acttcgtccg 20025200DNAArtificialPrimer
25tcggggagcg tcccggaccg cgagaggaga gagacgacgc catcgtcgag ccgggcgcac
60ggcggcgagc tcagcgatct ggagtcggat ctggtggggt ggcagaagac tggctgctcg
120tcgtcgccgg cgacaacaac gtcggctgcg gagctgatcg tgccgccagc
acaggagatc 180caggaattct tcgcggccgc 20026183DNAArtificialPrimer
26cactcttccc accgcagggt gaaagctcct cctgtgcacg ccctcccaag ttcaacggag
60atgaacgagt acttcgctgc tgaacagcga cgccaacaac aggctttcat tgacaagtac
120aactttgatc ctgtaaatga ctgccctctc ccaggcaggt ttgaatgggt
gaagctagac 180tga 18327804DNAZea mays 27accgtcttcg gtacgcgctc
actccgccct ctgcctttgt tactgccacg tttctctgaa 60tgctctcttg tgtggtgatt
gctgagagtg gtttagctgg atctagaatt acactctgaa 120atcgtgttct
gcctgtgctg attacttgcc gtcctttgta gcagcaaaat atagggacat
180ggtagtacga aacgaagata gaacctacac agcaatacga gaaatgtgta
atttggtgct 240tagcggtatt tatttaagca catgttggtg ttatagggca
cttggattca gaagtttgct 300gttaatttag gcacaggctt catactacat
gggtcaatag tatagggatt catattatag 360gcgatactat aataatttgt
tcgtctgcag agcttattat ttgccaaaat tagatattcc 420tattctgttt
ttgtttgtgt gctgttaaat tgttaacgcc tgaaggaata aatataaatg
480acgaaatttt gatgtttatc tctgctcctt tattgtgacc ataagtcaag
atcagatgca 540cttgttttaa atattgttgt ctgaagaaat aagtactgac
agtattttga tgcattgatc 600tgcttgtttg ttgtaacaaa atttaaaaat
aaagagtttc ctttttgttg ctctccttac 660ctcctgatgg tatctagtat
ctaccaactg acactatatt gcttctcttt acatacgtat 720cttgctcgat
gccttctccc tagtgttgac cagtgttact cacatagtct ttgctcattt
780cattgtaatg cagataccaa gcgg 8042844DNAArtificialPrimer
28gcaaggcctg cagcaactcg acgacttccc gccgcagaac ggag
442950DNAArtificialPrimer 29cgtcgagttg ctggttttac ttttagttct
agcagtctag cctcgcccat 503045DNAArtificialPrimer 30atgccggcgc
ccgcaatgtc gctctgcctc gcccactcat atcga 453134DNAArtificialPrimer
31gctagatcta gagcgacatt gcgggcgccg gcat 343238DNAArtificialPrimer
32ggctggatct cgtggacctt aaccgggccg cactcgga
383329DNAArtificialPrimer 33gctggatcct cagtctagct tcacccact
293429DNAArtificialPrimer 34gctagatctt cagtctagct tcacccact
293535DNAArtificialPrimer 35gctggatcct gcagggcctc gcccactcat atcga
353633DNAArtificialPrimer 36gctagatcta ccgtcttcgg tacgcgctca ctc
333729DNAArtificialPrimer 37gctggatccc cgcttggtat ctgcattac
293830DNAArtificialPrimer 38gcaccatggc cagccagacg ccgtcgccgt
303944DNAArtificialPrimer 39cgccgtgcgc ccggctcgat ggttaccacc
tccgaccccc cgtc 444043DNAArtificialPrimer 40cgcggtccgg gacgctcccc
gacggacgaa gtcgaagttg tac 434131DNAArtificialPrimer 41gctagatctt
cggggagcgt cccggaccgc g 314243DNAArtificialPrimer 42caccctgcgg
tgggaagagt ggcggccgcg aagaattcct gga 434341DNAArtificialPrimer
43gagcgcgtac cgaagacggt tcagtctagc ttcacccatt c 4144105DNAZea mays
44caggattccc cttgcgactg caccacgccc cacccaaacc cacctaccgc ctccgctccc
60ctctccagcg agtcgggggc acggacggga ccagacgacc aggcc 10545363DNAZea
mays 45attccccacc cagcacaaga gcaccattga tctgattgat atgtgtctac
cacaccacaa 60tgtgactagc tccgccgccg ccgccgtgga ccgtggagga ggcgcaacca
actgtggatc 120tcgatcgcat tagctttgtt gtctgttgta aaaattagag
tagttagctt tgtagccgga 180tgaatgatct tgtgtaacca acaggcgtgg
ttcacccctg gtcacacaac ctcactgtaa 240cctgttaact gctcaagctg
ctgtaaccga accaatctca tgtacgtagg cctagcgcag 300tcttctcatc
tgttcgtagc tctcgaaatt gattgaatgg aattagagtt aaaatttgca 360tgg
36346115DNAZea mays 46cccacaccca cctacctacc tacctaccac caacgcctcg
gctcccctct ccagcgagcc 60gcgaccgcgc ggcaggggaa gcttagcacg gacgggacca
gctgacgacc aggcc 11547340DNAZea mays 47attcctccaa gcacaagagc
accatttatc tgatgtgtct accacatgac tagcgccgcc 60gccgccgccg aggaggcgta
atcaactgga tcgcattagc tttcttgcct gttgtaaaaa 120ttagagtagt
tagcctgtag ttgaatggtc ctgtgtaacg aaacaggcgt ggttctgagt
180tacaccccga tccccaacct cactgtaacc gtttgactgc tcgctcaagc
tgtagccgaa 240ccatccatct caagtagcgt aggcctagcg tattcatctg
ttcctagttc tagttcttga 300atggattaat cgaatgagat cattgttaga
atctgcatgg 34048196DNAZea mays 48ctgccagatc caatccaaga ggtcgcctcg
tgtcctctcc tgtctgtctc tacttctctt 60gtaaaagtcg cttactaccc gtgtaacgct
tcgcttagct gtaactaaat tatgctcacg 120agatgggatt aatcatgtga
aggcccaacc ttgtacgtta gtggtgtgcg gttaacgtgt 180ccttccttag tccaga
19649217DNAZea mays 49gcgcgtaata cgactcacta tagggcgaat tgggtaccct
cgaggccggc cgggtctccc 60tcgttatttc cgggctccct cctgtgtaca ccactcccgc
cccgcccacc attttatccc 120cgcctctcct ggcctctgcc gccccgtcgc
acagaatcgc ttggtgcacc ctgcgagggc 180ctcctcgaaa ccctagcttg
cccagcccct ccgggcc 21750338DNAZea mays 50tggattcaga gggacgagag
agcagcaggc atggaatgga actcaccccc cgctccctcc 60acaccacccc agcgttgtgg
cagaggcgca taccgtcgtg ttagcttcgt ttctgctgta 120aaaaaaaccc
ttagtgtttt atttagcatg tagccttaac tggtcgtgtg ttacagtaca
180gaactgatgc tgagttacaa caccctgatc tgatccctca actccaatgt
aacccttaac 240agctcattct gtaaggaacc tgtcaccctg ttacctgttg
ctgaactaat gaagtagagc 300tagataatga cgttttatcg tagttattat taataact
33851120DNAZea mays 51tggtacgcct gcaggtaccg gtccggaatt cccgggtcga
cccacgcgtc cggccacggc 60accgcaccac acgcagagcg gcagaggcac accagcagcg
caaccggccg cgcgtgatcg 12052201DNAZea mays 52acccgtcaca gtccgttcgt
taacctcaag gtcaagcagc tagcaatgct ctcctccgac 60caccctcagc tggcaaactt
caaactggag tcatcactca tcagtagtgc tgattattat 120cctcccctga
tgtcgtacgt tatttttttg tagactgtca ctcatccatc actacccctt
180aattagcttt attattagcg t 20153191DNAZea mays 53tgcaggtacc
ggtccggaat tcccgggtcg acccacgcgt ccggtctgtg tgtttgtgat 60agaatccaaa
gacgcaagcg gctgcaggca gcagcgccgc gcaggcgttg tggcctgtgg
120gagaggaaaa agagaaagag gaaccggcca agacaagcaa gcgagaggcc
agggccgcgg 180cgttgcgtca g 19154333DNAZea mays 54agcgagcgtg
cgtccggtgc aaggtgaagc tagaaagaga aaagatgccc ccaacaaaca 60taacggagaa
gagaaaaacc aaacaattaa gcagctttat agcctaagct aaccaccacc
120attcatctcg tccaaatgcc ttgcttttct ctggagctag caggagcgcg
tagttattta 180gtactacttt acttattcag aggttatctt gaccccgata
gatcaatccg cttactgtgt 240aatttgtctc atgcatctct tagatggagt
ttaatcgtct taatttactg tacagcagct 300tgctggcttg caaagaaaga
tctggtttgt ctc 33355191DNAZea mays 55tgcaggtacc ggtccggaat
tcccgggtcg acccacgcgt ccggtctgtg tgtttgtgat 60agaatccaaa gacgcaagcg
gctgcaggca gcagcgccgc gcaggcgttg tggcctgtgg 120gagaggaaaa
agagaaagag gaaccggcca agacaagcaa gcgagaggcc agggccgcgg
180cgttgcgtca g 19156333DNAZea mays 56agcgagcgtg cgtccggtgc
aaggtgaagc tagaaagaga aaagatgccc ccaacaaaca 60taacggagaa gagaaaaacc
aaacaattaa gcagctttat agcctaagct aaccaccacc 120attcatctcg
tccaaatgcc ttgcttttct ctggagctag caggagcgcg tagttattta
180gtactacttt acttattcag aggttatctt gaccccgata gatcaatccg
cttactgtgt 240aatttgtctc atgcatctct tagatggagt ttaatcgtct
taatttactg tacagcagct 300tgctggcttg caaagaaaga tctggtttgt ctc
33357182DNAZea mays 57ggtaccggtc cggaattccc gggtcgaccc acgcgtccga
gaatccaaag cgcaagcggc 60tgcagcctgc aggcagcgcc gcgcaggcgt gggagtggcc
gagtgggagt gggagtgaaa 120aagaggaacc ggccaagaga agcaagcgag
aagaaggcag tgctgcggcg gcgttccgta 180ag 18258300DNAZea mays
58tagattcaga ggacatgaga gcagcagcat ggaactcacc tccgctccct ccaccgccgc
60agcgtcgtgg cagaggcgca taccatcgtg ttagctttgt ttctgttgta aaaacttagc
120gttagcttgt agccttaatt gtcgcgtgtc acagtacaga actgatgctg
agttacagca 180ccctgatatg atctggtccc tcaactccaa tgtaaccctt
aacagctcat tctgtaagga 240acctatcatc ctgttaccag ttgccgaatt
aatgaagtag agctagataa tgatgttctg 3005946PRTArtificialconsensus
59Pro Thr Thr Ala Glu Ile Glu Asp Phe Phe Ser Glu Ala Glu Glu Gln1
5 10 15Gln Gln Lys Gln Phe Ile Glu Lys Tyr Asn Phe Asp Ile Val Asn
Asp 20 25 30Glu Pro Leu Glu Gly Arg Tyr Glu Trp Val Lys Leu Lys Pro
35 40 45
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