U.S. patent application number 13/528367 was filed with the patent office on 2012-12-27 for modulating plant oil levels.
This patent application is currently assigned to CERES, INC.. Invention is credited to Steven Craig Bobzin, Boris Jankowski, Daniel Mumenthaler, Joel Cruz Rarang.
Application Number | 20120331583 13/528367 |
Document ID | / |
Family ID | 38327723 |
Filed Date | 2012-12-27 |
United States Patent
Application |
20120331583 |
Kind Code |
A1 |
Bobzin; Steven Craig ; et
al. |
December 27, 2012 |
MODULATING PLANT OIL LEVELS
Abstract
Methods and materials for modulating (e.g., increasing or
decreasing) oil levels in plants are disclosed. For example,
nucleic acids encoding oil-modulating polypeptides are disclosed as
well as methods for using such nucleic acids to transform plant
cells. Also disclosed are plants having increased oil levels and
plant products produced from plants having increased oil
levels.
Inventors: |
Bobzin; Steven Craig;
(Malibu, CA) ; Mumenthaler; Daniel; (Bonita,
CA) ; Jankowski; Boris; (Newbury Park, CA) ;
Rarang; Joel Cruz; (Granada Hills, CA) |
Assignee: |
CERES, INC.
Thousand Oaks
CA
|
Family ID: |
38327723 |
Appl. No.: |
13/528367 |
Filed: |
June 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12161935 |
Apr 15, 2009 |
8222482 |
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PCT/US07/02214 |
Jan 26, 2007 |
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13528367 |
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60762422 |
Jan 26, 2006 |
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60797077 |
May 1, 2006 |
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Current U.S.
Class: |
800/281 ;
426/615; 426/629; 426/630; 426/635; 435/412; 435/415; 435/419;
536/23.6; 554/9; 800/298 |
Current CPC
Class: |
C12N 15/8247 20130101;
C07K 14/415 20130101 |
Class at
Publication: |
800/281 ;
435/419; 435/415; 435/412; 800/298; 554/9; 536/23.6; 426/630;
426/635; 426/629; 426/615 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 5/10 20060101 C12N005/10; A01H 5/10 20060101
A01H005/10; A23L 1/36 20060101 A23L001/36; C12N 15/29 20060101
C12N015/29; A23K 1/00 20060101 A23K001/00; A23L 1/10 20060101
A23L001/10; C12N 15/82 20060101 C12N015/82; C11B 1/10 20060101
C11B001/10 |
Claims
1. A method of modulating the level of oil in a plant, said method
comprising introducing into a plant cell an isolated nucleic acid
comprising a nucleotide sequence encoding a polypeptide having 80
percent or greater sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs:81-92, SEQ ID
NO:94, SEQ ID NOs:96-107, SEQ ID NO:109, SEQ ID NOs:111-116, SEQ ID
NOs:118-134, SEQ ID NOs: 137-140, SEQ ID NO:142, SEQ ID NO:144, SEQ
ID NOs:146-147, SEQ ID NOs:149-150, SEQ ID NO:152, SEQ ID NO:154,
SEQ ID NOs:156-157, SEQ ID NOs:159-169, SEQ ID NOs:171-172, SEQ ID
NO:174, SEQ ID NO:176, SEQ ID NOs:178-179, SEQ ID NOs:181-182, SEQ
ID NOs:184-191, SEQ ID NOs:193-196, SEQ ID NOs:198-217, SEQ ID
NOs:219-220, SEQ ID NOs:222-229, SEQ ID NOs:231-238, SEQ ID NO:240,
SEQ ID NOs:242-250, SEQ ID NOs:252-330, SEQ ID NOs:332-333, SEQ ID
NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID
NO:346-348, SEQ ID NOs:350-357, SEQ ID NOs:359-366, SEQ ID NO:368,
SEQ ID NOs:370-372, SEQ ID NOs:374-376, SEQ ID NOs:378-379, SEQ ID
NOs:381-396, SEQ ID NO:398, and SEQ ID NOs:502-545, wherein a
tissue of a plant produced from said plant cell has a difference in
the level of oil as compared to the corresponding level in tissue
of a control plant that does not comprise said nucleic acid.
2. The method of claim 1, said nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID
NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ ID NO:109, SEQ ID
NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID NOs:120-121, SEQ
ID NOs:125-132, SEQ ID NO:134, SEQ ID NOs:139-140, SEQ ID NO:142,
SEQ ID NO:144, SEQ ID NOs:146-147, SEQ ID NO:149, SEQ ID NO:152,
SEQ ID NO:154, SEQ ID NOs:156-157, SEQ ID NO:159, SEQ ID
NOs:162-168, SEQ ID NOs:171-172, SEQ ID NO:174, SEQ ID NO:176, SEQ
ID NO:178, SEQ ID NO:181, SEQ ID NO:184, SEQ ID NOs:193-194, SEQ ID
NO:198, SEQ ID NO:207, SEQ ID NOs:215-217, SEQ ID NOs:219-220, SEQ
ID NOs:222-223, SEQ ID NOs:231-232, SEQ ID NO:235, SEQ ID NO:238,
SEQ ID NO:240, SEQ ID NO:242, SEQ ID NOs:244-246, SEQ ID NO:250,
SEQ ID NO:252, SEQ ID NO:254, SEQ ID NOs:257-258, SEQ ID NO:261,
SEQ ID NOs:265-267, SEQ ID NO:270, SEQ ID NO:279, SEQ ID
NOs:287-293, SEQ ID NOs:300-301, SEQ ID NO:304, SEQ ID NOs:307-309,
SEQ ID NOs:311-313, SEQ ID NOs:317-318, SEQ ID NO:320, SEQ ID
NOs:323-324, SEQ ID NO:327, SEQ ID NO:332, SEQ ID NO:335, SEQ ID
NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NOs:346-348, SEQ
ID NOs:350-351, SEQ ID NOs:355-357, SEQ ID NOs:359-361, SEQ ID
NOs:363-366, SEQ ID NO:368, SEQ ID NOs:370-372, SEQ ID NO:374, SEQ
ID NO:378, SEQ ID NO:381, SEQ ID NO:398, SEQ ID NOs:502-505, SEQ ID
NOs:507-519, SEQ ID NO:521, SEQ ID NOs:524-527, SEQ ID NOs:529-534,
SEQ ID NO:541, and SEQ ID NOs:544-545, wherein a tissue of a plant
produced from said plant cell has a difference in the level of oil
as compared to the corresponding level in tissue of a control plant
that does not comprise said nucleic acid.
3. (canceled)
4. The method of claim 1, wherein said sequence identity is 85
percent or greater.
5. The method of claim 4, wherein said sequence identity is 90
percent or greater.
6. The method of claim 4, wherein said sequence identity is 95
percent or greater.
7. The method of claim 1, wherein said nucleotide sequence encodes
a polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:81, SEQ ID NO:94, SEQ ID NO:111, SEQ
ID NO:152, SEQ ID NO:159, SEQ ID NO:171, SEQ ID NO:176, SEQ ID
NO:178, SEQ ID NO:193, SEQ ID NO:332, SEQ ID NO:342, SEQ ID NO:344,
SEQ ID NO:346, SEQ ID NO:359, SEQ ID NO:374, and SEQ ID NO:398.
8. The method of claim 1, wherein said difference is an increase in
the level of oil.
9. The method of claim 1, wherein said isolated nucleic acid is
operably linked to a regulatory region.
10. The method of claim 9, wherein said regulatory region is a
tissue-preferential promoter or a broadly expressing promoter.
11. (canceled)
12. (canceled)
13. The method of claim 1, wherein said plant is a dicot.
14. The method of claim 13, wherein said plant is a member of the
genus Anacardium, Arachis, Azadirachta, Brassica, Cannabis,
Carthamus, Corylus, Crambe, Cucurbita, Glycine, Gossypium,
Helianthus, Jatropha, Juglans, Linum, Olea, Papaver, Persea,
Prunus, Ricinus, Sesamum, Simmondsia, or Vitis.
15. The method of claim 1, wherein said plant is a monocot.
16. The method of claim 15, wherein said plant is a member of the
genus Cocos, Elaeis, Oryza, or Zea.
17. The method of claim 1, wherein said tissue is seed tissue.
18. A method of producing a plant tissue, said method comprising
growing a plant cell comprising an exogenous nucleic acid
comprising a nucleotide sequence encoding a polypeptide having 80
percent or greater sequence identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs:81-92, SEQ ID
NO:94, SEQ ID NOs:96-107, SEQ ID NO:109, SEQ ID NOs:111-116, SEQ ID
NOs:118-134, SEQ ID NOs: 137-140, SEQ ID NO:142, SEQ ID NO:144, SEQ
ID NOs:146-147, SEQ ID NOs:149-150, SEQ ID NO:152, SEQ ID NO:154,
SEQ ID NOs:156-157, SEQ ID NOs:159-169, SEQ ID NOs:171-172, SEQ ID
NO:174, SEQ ID NO:176, SEQ ID NOs:178-179, SEQ ID NOs:181-182, SEQ
ID NOs:184-191, SEQ ID NOs:193-196, SEQ ID NOs:198-217, SEQ ID
NOs:219-220, SEQ ID NOs:222-229, SEQ ID NOs:231-238, SEQ ID NO:240,
SEQ ID NOs:242-250, SEQ ID NOs:252-330, SEQ ID NOs:332-333, SEQ ID
NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID
NO:346-348, SEQ ID NOs:350-357, SEQ ID NOs:359-366, SEQ ID NO:368,
SEQ ID NOs:370-372, SEQ ID NOs:374-376, SEQ ID NOs:378-379, SEQ ID
NOs:381-396, SEQ ID NO:398, and SEQ ID NOs:502-545, wherein said
tissue has a difference in the level of oil as compared to the
corresponding level in tissue of a control plant that does not
comprise said nucleic acid.
19. The method of claim 18, said nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID
NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ ID NO:109, SEQ ID
NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID NOs:120-121, SEQ
ID NOs:125-132, SEQ ID NO:134, SEQ ID NOs:139-140, SEQ ID NO:142,
SEQ ID NO:144, SEQ ID NOs:146-147, SEQ ID NO:149, SEQ ID NO:152,
SEQ ID NO:154, SEQ ID NOs:156-157, SEQ ID NO:159, SEQ ID
NOs:162-168, SEQ ID NOs:171-172, SEQ ID NO:174, SEQ ID NO:176, SEQ
ID NO:178, SEQ ID NO:181, SEQ ID NO:184, SEQ ID NOs:193-194, SEQ ID
NO:198, SEQ ID NO:207, SEQ ID NOs:215-217, SEQ ID NOs:219-220, SEQ
ID NOs:222-223, SEQ ID NOs:231-232, SEQ ID NO:235, SEQ ID NO:238,
SEQ ID NO:240, SEQ ID NO:242, SEQ ID NOs:244-246, SEQ ID NO:250,
SEQ ID NO:252, SEQ ID NO:254, SEQ ID NOs:257-258, SEQ ID NO:261,
SEQ ID NOs:265-267, SEQ ID NO:270, SEQ ID NO:279, SEQ ID
NOs:287-293, SEQ ID NOs:300-301, SEQ ID NO:304, SEQ ID NOs:307-309,
SEQ ID NOs:311-313, SEQ ID NOs:317-318, SEQ ID NO:320, SEQ ID
NOs:323-324, SEQ ID NO:327, SEQ ID NO:332, SEQ ID NO:335, SEQ ID
NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NOs:346-348, SEQ
ID NOs:350-351, SEQ ID NOs:355-357, SEQ ID NOs:359-361, SEQ ID
NOs:363-366, SEQ ID NO:368, SEQ ID NOs:370-372, SEQ ID NO:374, SEQ
ID NO:378, SEQ ID NO:381, SEQ ID NO:398, SEQ ID NOs:502-505, SEQ ID
NOs:507-519, SEQ ID NO:521, SEQ ID NOs:524-527, SEQ ID NOs:529-534,
SEQ ID NO:541, and SEQ ID NOs:544-545, wherein said tissue has a
difference in the level of oil as compared to the corresponding
level in tissue of a control plant that does not comprise said
nucleic acid.
20. (canceled)
21. The method of claim 18, wherein said sequence identity is 85
percent or greater.
22. The method of claim 21, wherein said sequence identity is 90
percent or greater.
23. The method of claim 21, wherein said sequence identity is 95
percent or greater.
24. The method of claim 18, wherein said nucleotide sequence
encodes a polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:81, SEQ ID NO:94, SEQ ID
NO:111, SEQ ID NO:152, SEQ ID NO:159, SEQ ID NO:171, SEQ ID NO:176,
SEQ ID NO:178, SEQ ID NO:193, SEQ ID NO:332, SEQ ID NO:342, SEQ ID
NO:344, SEQ ID NO:346, SEQ ID NO:359, SEQ ID NO:374, and SEQ ID
NO:398.
25. The method of claim 18, wherein said difference is an increase
in the level of oil.
26. The method of claim 18, wherein said exogenous nucleic acid is
operably linked to a regulatory region.
27. The method of claim 26, wherein said regulatory region is a
tissue-preferential promoter or a broadly expressing promoter.
28. (canceled)
29. (canceled)
30. The method of claim 18, wherein said plant tissue is
dicotyledonous.
31. The method of claim 30, wherein said plant tissue is a member
of the genus Anacardium, Arachis, Azadirachta, Brassica, Cannabis,
Carthamus, Corylus, Crambe, Cucurbita, Glycine, Gossypium,
Helianthus, Jatropha, Juglans, Linum, Olea, Papaver, Persea,
Prunus, Ricinus, Sesamum, Simmondsia, or Vitis.
32. The method of claim 18, wherein said plant tissue is
monocotyledonous.
33. The method of claim 32, wherein said plant tissue is a member
of the genus Cocos, Elaeis, Oryza, or Zea.
34. The method of claim 18, wherein said tissue is seed tissue.
35. A plant cell comprising an exogenous nucleic acid comprising a
nucleotide sequence encoding a polypeptide having 80 percent or
greater sequence identity to an amino acid sequence selected from
the group consisting of SEQ ID NOs:81-92, SEQ ID NO:94, SEQ ID
NOs:96-107, SEQ ID NO:109, SEQ ID NOs:111-116, SEQ ID NOs:118-134,
SEQ ID NOs: 137-140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID
NOs:146-147, SEQ ID NOs:149-150, SEQ ID NO:152, SEQ ID NO:154, SEQ
ID NOs:156-157, SEQ ID NOs:159-169, SEQ ID NOs:171-172, SEQ ID
NO:174, SEQ ID NO:176, SEQ ID NOs:178-179, SEQ ID NOs:181-182, SEQ
ID NOs:184-191, SEQ ID NOs:193-196, SEQ ID NOs:198-217, SEQ ID
NOs:219-220, SEQ ID NOs:222-229, SEQ ID NOs:231-238, SEQ ID NO:240,
SEQ ID NOs:242-250, SEQ ID NOs:252-330, SEQ ID NOs:332-333, SEQ ID
NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID
NO:346-348, SEQ ID NOs:350-357, SEQ ID NOs:359-366, SEQ ID NO:368,
SEQ ID NOs:370-372, SEQ ID NOs:374-376, SEQ ID NOs:378-379, SEQ ID
NOs:381-396, SEQ ID NO:398, and SEQ ID NOs: 502-545, wherein a
tissue of a plant produced from said plant cell has a difference in
the level of oil as compared to the corresponding level in tissue
of a control plant that does not comprise said nucleic acid.
36. The plant cell of claim 35, said nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID
NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ ID NO:109, SEQ ID
NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID NOs:120-121, SEQ
ID NOs:125-132, SEQ ID NO:134, SEQ ID NOs:139-140, SEQ ID NO:142,
SEQ ID NO:144, SEQ ID NOs:146-147, SEQ ID NO:149, SEQ ID NO:152,
SEQ ID NO:154, SEQ ID NOs:156-157, SEQ ID NO:159, SEQ ID
NOs:162-168, SEQ ID NOs:171-172, SEQ ID NO:174, SEQ ID NO:176, SEQ
ID NO:178, SEQ ID NO:181, SEQ ID NO:184, SEQ ID NOs:193-194, SEQ ID
NO:198, SEQ ID NO:207, SEQ ID NOs:215-217, SEQ ID NOs:219-220, SEQ
ID NOs:222-223, SEQ ID NOs:231-232, SEQ ID NO:235, SEQ ID NO:238,
SEQ ID NO:240, SEQ ID NO:242, SEQ ID NOs:244-246, SEQ ID NO:250,
SEQ ID NO:252, SEQ ID NO:254, SEQ ID NOs:257-258, SEQ ID NO:261,
SEQ ID NOs:265-267, SEQ ID NO:270, SEQ ID NO:279, SEQ ID
NOs:287-293, SEQ ID NOs:300-301, SEQ ID NO:304, SEQ ID NOs:307-309,
SEQ ID NOs:311-313, SEQ ID NOs:317-318, SEQ ID NO:320, SEQ ID
NOs:323-324, SEQ ID NO:327, SEQ ID NO:332, SEQ ID NO:335, SEQ ID
NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NOs:346-348, SEQ
ID NOs:350-351, SEQ ID NOs:355-357, SEQ ID NOs:359-361, SEQ ID
NOs:363-366, SEQ ID NO:368, SEQ ID NOs:370-372, SEQ ID NO:374, SEQ
ID NO:378, SEQ ID NO:381, SEQ ID NO:398, SEQ ID NOs:502-505, SEQ ID
NOs:507-519, SEQ ID NO:521, SEQ ID NOs:524-527, SEQ ID NOs:529-534,
SEQ ID NO:541, and SEQ ID NOs:544-545, wherein a tissue of a plant
produced from said plant cell has a difference in the level of oil
as compared to the corresponding level in tissue of a control plant
that does not comprise said nucleic acid.
37. (canceled)
38. The plant cell of claim 35, wherein said sequence identity is
85 percent or greater.
39. The plant cell of claim 38, wherein said sequence identity is
90 percent or greater.
40. The plant cell of claim 38, wherein said sequence identity is
95 percent or greater.
41. The plant cell of claim 37, wherein said nucleotide sequence
encodes a polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:81, SEQ ID NO:94, SEQ ID
NO:111, SEQ ID NO:152, SEQ ID NO:159, SEQ ID NO:171, SEQ ID NO:176,
SEQ ID NO:178, SEQ ID NO:193, SEQ ID NO:332, SEQ ID NO:342, SEQ ID
NO:344, SEQ ID NO:346, SEQ ID NO:359, SEQ ID NO:374, and SEQ ID
NO:398.
42. The plant cell of claim 35, wherein said difference is an
increase in the level of oil.
43. The plant cell of claim 35, wherein said exogenous nucleic acid
is operably linked to a regulatory region.
44. The plant cell of claim 43, wherein said regulatory region is a
tissue-preferential promoter or a broadly expressing promoter.
45. (canceled)
46. (canceled)
47. The plant cell of claim 35, wherein said plant is a dicot.
48. The plant cell of claim 47, wherein said plant is a member of
the genus Anacardium, Arachis, Azadirachta, Brassica, Cannabis,
Carthamus, Corylus, Crambe, Cucurbita, Glycine, Gossypium,
Helianthus, Jatropha, Juglans, Linum, Olea, Papaver, Persea,
Prunus, Ricinus, Sesamum, Simmondsia, or Vitis.
49. The plant cell of claim 35, wherein said plant is a
monocot.
50. The plant cell of claim 49, wherein said plant is a member of
the genus Cocos, Elaeis, Oryza, or Zea.
51. The plant cell of claim 35, wherein said tissue is seed
tissue.
52. A transgenic plant comprising the plant cell of claim 35.
53. Progeny of the plant of claim 52, wherein said progeny has a
difference in the level of oil as compared to the level of oil in a
corresponding control plant that does not comprise said exogenous
nucleic acid.
54. Seed from a transgenic plant according to claim 52.
55. Vegetative tissue from a transgenic plant according to claim
52.
56. A food or feed product comprising seed or vegetative tissue
from a transgenic plant according to claim 52.
57. (canceled)
58. Oil from the seed of claim 54.
59. A method of making oil, said method comprising extracting oil
from the seed of claim 54.
60. An isolated nucleic acid comprising a) a nucleotide sequence
having 95% or greater sequence identity to a nucleotide sequence
selected from the group consisting of SEQ ID NO:95, SEQ ID NO:108,
SEQ ID NO:117, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID
NO:148, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:173, SEQ ID NO:180,
SEQ ID NO:183, SEQ ID NO:197, SEQ ID NO:218, SEQ ID NO:221, SEQ ID
NO:230, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:251, SEQ ID NO:334,
SEQ ID NO:336, SEQ ID NO:349, SEQ ID NO:367, SEQ ID NO:369, SEQ ID
NO:377, SEQ ID NO:380, SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476,
SEQ ID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480, SEQ ID
NO:481, SEQ ID NO:482, SEQ ID NO:483, SEQ ID NO:484, SEQ ID NO:485,
SEQ ID NO:486, SEQ ID NO:487, SEQ ID NO:488, SEQ ID NO:489, SEQ ID
NO:490, SEQ ID NO:491, SEQ ID NO:492, SEQ ID NO:493, SEQ ID NO:494,
SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:498, SEQ ID
NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:546, SEQ ID NO:547,
SEQ ID NO:549, SEQ ID NO:551, SEQ ID NO:553, SEQ ID NO:555, SEQ ID
NO:556, SEQ ID NO:557, SEQ ID NO:559, SEQ ID NO:561, SEQ ID NO:562,
SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID
NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573,
and SEQ ID NO:575; or b) a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NO:96,
SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:142, SEQ ID NO:144, SEQ ID
NO:146, SEQ ID NO:149, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:174,
SEQ ID NO:181, SEQ ID NO:184, SEQ ID NO:198, SEQ ID NO:219, SEQ ID
NO:222, SEQ ID NO:231, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:252,
SEQ ID NO:270, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:300, SEQ ID
NO:308, SEQ ID NO:309, SEQ ID NO:318, SEQ ID NO:335, SEQ ID NO:337,
SEQ ID NO:350, SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:378, SEQ ID
NO:381, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505,
SEQ ID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ ID NO:513, SEQ ID
NO:514, SEQ ID NO:515, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:521,
SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:529, SEQ ID
NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:541,
and SEQ ID NO:545.
61. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
12/161,935, filed on Apr. 15, 2009, which is a National Stage
application under 35 U.S.C. .sctn.371 of International Application
No. PCT/US2007/002214, filed Jan. 26, 2007, which claims priority
under 35 U.S.C. 119(e) to U.S. Provisional Application No.
60/762,422, filed Jan. 26, 2006, and U.S. Provisional Application
No. 60/797,077, filed May 1, 2006, the disclosures of which are
incorporated herein by reference in their entirety.
BACKGROUND
[0002] 1. Technical Field
[0003] This document relates to methods and materials involved in
modulating (e.g., increasing or decreasing) oil levels in plants.
For example, this document provides plants having increased oil
levels as well as materials and methods for making plants and plant
products having increased oil levels.
[0004] 2. Incorporation-By-Reference & Texts
[0005] The material in the accompanying sequence listing is hereby
incorporated by reference into this application. The accompanying
file, named 11696204WO1Sequence.txt, was created on Jan. 26, 2007
and is 1128 KB. The file can be accessed using Microsoft Word on a
computer that uses Windows OS.
[0006] 3. Background Information
[0007] Fat, protein, and carbohydrates are nutrients that supply
calories to the body. Fat provides nine calories per gram, which is
more than twice the number provided by carbohydrates or protein.
Dietary fats are composed of fatty acids and glycerol. The glycerol
can be converted to glucose by the liver and used as a source of
energy. The fatty acids are a good source of energy for many
tissues, especially heart and skeletal muscle.
[0008] Fatty acids consist of carbon chains of various lengths and
a terminal carboxylic acid group. Saturated fatty acids do not
contain any double bonds or other functional groups along the
chain. A saturated fatty acid has the maximum possible number of
hydrogen atoms attached to every carbon atom. Therefore, it is said
to be saturated with hydrogen atoms. Eating too much saturated fat
is one of the major risk factors for heart disease. Saturated fats
are found in animal products such as butter, cheese, whole milk,
ice cream, cream, and fatty meats. Saturated fats are also found in
some vegetable oils, such as coconut, palm, and palm kernel oils.
Most other vegetable oils contain unsaturated fat that helps to
lower blood cholesterol if used in place of saturated fat.
[0009] Unsaturated fatty acids contain one or more double bonds
between carbon atoms and, therefore, two fewer hydrogen atoms per
double bond. A fatty acid with a single double bond is called a
monounsaturated fatty acid. A fatty acid with two or more double
bonds is called a polyunsaturated fatty acid. Polyunsaturated fats
are liquid at room temperature, and remain in liquid form even when
refrigerated or frozen. Polyunsaturated fats are divided into two
families: the omega-3 fats and the omega-6 fats.
[0010] The omega-3 family of fatty acids includes alpha-linolenic
acid (ALA). ALA is an essential fatty acid that cannot be
synthesized in the body and must, therefore, be consumed in the
diet. Dietary sources of ALA include canola, flaxseed, flaxseed
oil, soybean, and pumpkin seed oil. Omega-3 fatty acids have been
found to reduce the risks of heart problems, lower high blood
pressure, and ameliorate autoimmune diseases.
[0011] Omega-6 fatty acids are beneficial as well. The omega-6
family of fatty acids includes linoleic acid, which is another
essential fatty acid. The body converts linoleic acid to gamma
linoleic acid (GLA) and ultimately to prostaglandins, which are
hormone-like molecules that help regulate inflammation and blood
pressure as well as heart, gastrointestinal, and kidney functions.
The main sources of omega-6 fatty acids are vegetable oils such as
corn oil and soy oil.
[0012] Vegetable oil is fat extracted from plant sources. Vegetable
oils are used in cooking, in making margarine and other processed
foods, and in producing several non-food items such as soap,
cosmetics, medicine, and paint. Since vegetable oils are usually
extracted from the seeds of the plant, seed oil yield has a
significant impact on the economics of producing many products.
Increasing seed oil content may increase the economic return per
unit to the seller of the seed in addition to increasing the
nutritional value to the consumer of the seed.
SUMMARY
[0013] This document provides methods and materials related to
plants having modulated (e.g., increased or decreased) levels of
oil. For example, this document provides transgenic plants and
plant cells having increased levels of oil, nucleic acids used to
generate transgenic plants and plant cells having increased levels
of oil, and methods for making plants and plant cells having
increased levels of oil. Such plants and plant cells can be grown
to produce, for example, seeds having increased oil content.
Increasing the oil content of seeds can increase the nutritional
value of the seeds and the yield of oil obtained from the seeds,
which may benefit both food consumers and producers.
[0014] In one aspect, a method of modulating the level of oil in a
plant is provided. The method comprises introducing into a plant
cell an isolated nucleic acid comprising a nucleotide sequence
encoding a polypeptide having 80 percent or greater sequence
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs:81-92, SEQ ID NO:94, SEQ ID NOs:96-107,
SEQ ID NO:109, SEQ ID NOs:111-116, SEQ ID NOs:118-134, SEQ ID
NOs:136-140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NOs:146-147, SEQ
ID NOs:149-150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NOs:156-157,
SEQ ID NOs:159-169, SEQ ID NOs:171-172, SEQ ID NO:174, SEQ ID
NO:176, SEQ ID NOs:178-179, SEQ ID NOs:181-182, SEQ ID NOs:184-191,
SEQ ID NOs:193-196, SEQ ID NOs:198-217, SEQ ID NOs:219-220, SEQ ID
NOs:222-229, SEQ ID NOs:231-238, SEQ ID NO:240, SEQ ID NOs:242-250,
SEQ ID NOs:252-330, SEQ ID NOs:332-333, SEQ ID NO:335, SEQ ID
NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346-348, SEQ
ID NOs:350-357, SEQ ID NOs:359-366, SEQ ID NO:368, SEQ ID
NOs:370-372, SEQ ID NOs:374-376, SEQ ID NOs:378-379, SEQ ID
NOs:381-396, SEQ ID NO:398, SEQ ID NOs:502-545, and the consensus
sequences set forth in FIGS. 1-13, where a tissue of a plant
produced from the plant cell has a difference in the level of oil
as compared to the corresponding level in tissue of a control plant
that does not comprise the nucleic acid.
[0015] In another aspect, a method of modulating the level of oil
in a plant is provided. The method comprises introducing into a
plant cell an isolated nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ
ID NO:91, SEQ ID NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ
ID NO:109, SEQ ID NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID
NOs:120-121, SEQ ID NOs:125-132, SEQ ID NO:134, SEQ ID NO:136, SEQ
ID NOs:139-140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NOs:146-147,
SEQ ID NO:149, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NOs:156-157,
SEQ ID NO:159, SEQ ID NOs:162-168, SEQ ID NOs:171-172, SEQ ID
NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:184,
SEQ ID NOs:193-194, SEQ ID NO:198, SEQ ID NO:207, SEQ ID
NOs:215-217, SEQ ID NOs:219-220, SEQ ID NOs:222-223, SEQ ID
NOs:231-232, SEQ ID NO:235, SEQ ID NO:238, SEQ ID NO:240, SEQ ID
NO:242, SEQ ID NOs:244-246, SEQ ID NO:250, SEQ ID NO:252, SEQ ID
NO:254, SEQ ID NOs:257-258, SEQ ID NO:261, SEQ ID NOs:265-267, SEQ
ID NO:270, SEQ ID NO:279, SEQ ID NOs:287-293, SEQ ID NOs:300-301,
SEQ ID NO:304, SEQ ID NOs:307-309, SEQ ID NOs:311-313, SEQ ID
NOs:317-318, SEQ ID NO:320, SEQ ID NOs:323-324, SEQ ID NO:327, SEQ
ID NO:332, SEQ ID NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID
NO:344, SEQ ID NOs:346-348, SEQ ID NOs:350-351, SEQ ID NOs:355-357,
SEQ ID NOs:359-361, SEQ ID NOs:363-366, SEQ ID NO:368, SEQ ID
NOs:370-372, SEQ ID NO:374, SEQ ID NO:378, SEQ ID NO:381, SEQ ID
NO:398, SEQ ID NOs:502-505, SEQ ID NOs:507-519, SEQ ID NO:521, SEQ
ID NOs:524-527, SEQ ID NOs:529-534, SEQ ID NO:541, SEQ ID
NOs:544-545, and the consensus sequences set forth in FIGS. 1-13,
where a tissue of a plant produced from the plant cell has a
difference in the level of oil as compared to the corresponding
level in tissue of a control plant that does not comprise the
nucleic acid.
[0016] In another aspect, a method of modulating the level of oil
in a plant is provided. The method comprises introducing into a
plant cell an isolated nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ
ID NO:91, SEQ ID NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ
ID NO:109, SEQ ID NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID
NOs:120-121, SEQ ID NOs:125-132, SEQ ID NO:134, SEQ ID NO:136, SEQ
ID NOs:139-140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NOs:146-147,
SEQ ID NO:149, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NOs:156-157,
SEQ ID NO:159, SEQ ID NOs:162-168, SEQ ID NOs:171-172, SEQ ID
NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:184,
SEQ ID NOs:193-194, SEQ ID NO:198, SEQ ID NO:207, SEQ ID
NOs:215-217, SEQ ID NOs:219-220, SEQ ID NOs:222-223, SEQ ID
NOs:231-232, SEQ ID NO:235, SEQ ID NO:238, SEQ ID NO:240, SEQ ID
NO:242, SEQ ID NOs:244-246, SEQ ID NO:250, SEQ ID NO:252, SEQ ID
NO:254, SEQ ID NOs:257-258, SEQ ID NO:261, SEQ ID NOs:265-267, SEQ
ID NO:270, SEQ ID NO:279, SEQ ID NOs:287-293, SEQ ID NOs:300-301,
SEQ ID NO:304, SEQ ID NOs:307-309, SEQ ID NOs:311-313, SEQ ID
NOs:317-318, SEQ ID NO:320, SEQ ID NOs:323-324, SEQ ID NO:327, SEQ
ID NO:332, SEQ ID NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID
NO:344, SEQ ID NOs:346-348, SEQ ID NOs:350-351, SEQ ID NOs:355-357,
SEQ ID NOs:359-361, SEQ ID NOs:363-366, SEQ ID NO:368, SEQ ID
NOs:370-372, SEQ ID NO:374, SEQ ID NO:378, SEQ ID NO:381, SEQ ID
NO:398, SEQ ID NOs:502-505, SEQ ID NOs:507-519, SEQ ID NO:521, SEQ
ID NOs:524-527, SEQ ID NOs:529-534, SEQ ID NO:541, and SEQ ID
NOs:544-545, where a tissue of a plant produced from the plant cell
has a difference in the level of oil as compared to the
corresponding level in tissue of a control plant that does not
comprise the nucleic acid.
[0017] The sequence identity can be 85 percent or greater, 90
percent or greater, or 95 percent or greater. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:81. The nucleotide sequence can encode a
polypeptide comprising an amino acid sequence corresponding to SEQ
ID NO:94. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:111.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:136. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:152. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:159. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:171.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:176. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:178. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:193. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:332.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:342. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:344. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:346. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:359.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:374. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:398. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to a
consensus sequence set forth in FIG. 1, FIG. 2, FIG. 3, FIG. 4,
FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12,
or FIG. 13. The difference can be an increase in the level of oil.
The isolated nucleic acid can be operably linked to a regulatory
region. The regulatory region can be a tissue-preferential
regulatory region. tissue-preferential regulatory region can be a
promoter. The regulatory region can be a broadly expressing
promoter. The plant can be a dicot. The plant can be a member of
the genus Anacardium, Arachis, Azadirachta, Brassica, Cannabis,
Carthamus, Corylus, Crambe, Cucurbita, Glycine, Gossypium,
Helianthus, Jatropha, Juglans, Linum, Olea, Papaver, Persea,
Prunus, Ricinus, Sesamum, Simmondsia, or Vitis. The plant can be a
monocot. The plant can be a member of the genus Cocos, Elaeis,
Oryza, or Zea. The tissue can be seed tissue.
[0018] A method of producing a plant tissue is also provided. The
method comprises growing a plant cell comprising an exogenous
nucleic acid comprising a nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs:81-92, SEQ ID NO:94, SEQ ID NOs:96-107, SEQ ID NO:109, SEQ ID
NOs:111-116, SEQ ID NOs:118-134, SEQ ID NOs:136-140, SEQ ID NO:142,
SEQ ID NO:144, SEQ ID NOs:146-147, SEQ ID NOs:149-150, SEQ ID
NO:152, SEQ ID NO:154, SEQ ID NOs:156-157, SEQ ID NOs:159-169, SEQ
ID NOs:171-172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NOs:178-179,
SEQ ID NOs:181-182, SEQ ID NOs:184-191, SEQ ID NOs:193-196, SEQ ID
NOs:198-217, SEQ ID NOs:219-220, SEQ ID NOs:222-229, SEQ ID
NOs:231-238, SEQ ID NO:240, SEQ ID NOs:242-250, SEQ ID NOs:252-330,
SEQ ID NOs:332-333, SEQ ID NO:335, SEQ ID NOs:337-340, SEQ ID
NO:342, SEQ ID NO:344, SEQ ID NO:346-348, SEQ ID NOs:350-357, SEQ
ID NOs:359-366, SEQ ID NO:368, SEQ ID NOs:370-372, SEQ ID
NOs:374-376, SEQ ID NOs:378-379, SEQ ID NOs:381-396, SEQ ID NO:398,
SEQ ID NOs:502-545, and the consensus sequences set forth in FIGS.
1-13, where the tissue has a difference in the level of oil as
compared to the corresponding level in tissue of a control plant
that does not comprise the nucleic acid.
[0019] In another aspect, a method of producing a plant tissue is
provided. The method comprises growing a plant cell comprising an
exogenous nucleic acid comprising a nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID
NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ ID NO:109, SEQ ID
NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID NOs:120-121, SEQ
ID NOs:125-132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NOs:139-140,
SEQ ID NO:142, SEQ ID NO:144, SEQ ID NOs:146-147, SEQ ID NO:149,
SEQ ID NO:152, SEQ ID NO:154, SEQ ID NOs:156-157, SEQ ID NO:159,
SEQ ID NOs:162-168, SEQ ID NOs:171-172, SEQ ID NO:174, SEQ ID
NO:176, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:184, SEQ ID
NOs:193-194, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NOs:215-217, SEQ
ID NOs:219-220, SEQ ID NOs:222-223, SEQ ID NOs:231-232, SEQ ID
NO:235, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID
NOs:244-246, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID
NOs:257-258, SEQ ID NO:261, SEQ ID NOs:265-267, SEQ ID NO:270, SEQ
ID NO:279, SEQ ID NOs:287-293, SEQ ID NOs:300-301, SEQ ID NO:304,
SEQ ID NOs:307-309, SEQ ID NOs:311-313, SEQ ID NOs:317-318, SEQ ID
NO:320, SEQ ID NOs:323-324, SEQ ID NO:327, SEQ ID NO:332, SEQ ID
NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID
NOs:346-348, SEQ ID NOs:350-351, SEQ ID NOs:355-357, SEQ ID
NOs:359-361, SEQ ID NOs:363-366, SEQ ID NO:368, SEQ ID NOs:370-372,
SEQ ID NO:374, SEQ ID NO:378, SEQ ID NO:381, SEQ ID NO:398, SEQ ID
NOs:502-505, SEQ ID NOs:507-519, SEQ ID NO:521, SEQ ID NOs:524-527,
SEQ ID NOs:529-534, SEQ ID NO:541, SEQ ID NOs:544-545, and the
consensus sequences set forth in FIGS. 1-13, where the tissue has a
difference in the level of oil as compared to the corresponding
level in tissue of a control plant that does not comprise the
nucleic acid.
[0020] In another aspect, a method of producing a plant tissue is
provided. The method comprises growing a plant cell comprising an
exogenous nucleic acid comprising a nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ ID NO:91, SEQ ID
NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ ID NO:109, SEQ ID
NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID NOs:120-121, SEQ
ID NOs:125-132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NOs:139-140,
SEQ ID NO:142, SEQ ID NO:144, SEQ ID NOs:146-147, SEQ ID NO:149,
SEQ ID NO:152, SEQ ID NO:154, SEQ ID NOs:156-157, SEQ ID NO:159,
SEQ ID NOs:162-168, SEQ ID NOs:171-172, SEQ ID NO:174, SEQ ID
NO:176, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:184, SEQ ID
NOs:193-194, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NOs:215-217, SEQ
ID NOs:219-220, SEQ ID NOs:222-223, SEQ ID NOs:231-232, SEQ ID
NO:235, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID
NOs:244-246, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID
NOs:257-258, SEQ ID NO:261, SEQ ID NOs:265-267, SEQ ID NO:270, SEQ
ID NO:279, SEQ ID NOs:287-293, SEQ ID NOs:300-301, SEQ ID NO:304,
SEQ ID NOs:307-309, SEQ ID NOs:311-313, SEQ ID NOs:317-318, SEQ ID
NO:320, SEQ ID NOs:323-324, SEQ ID NO:327, SEQ ID NO:332, SEQ ID
NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID
NOs:346-348, SEQ ID NOs:350-351, SEQ ID NOs:355-357, SEQ ID
NOs:359-361, SEQ ID NOs:363-366, SEQ ID NO:368, SEQ ID NOs:370-372,
SEQ ID NO:374, SEQ ID NO:378, SEQ ID NO:381, SEQ ID NO:398, SEQ ID
NOs:502-505, SEQ ID NOs:507-519, SEQ ID NO:521, SEQ ID NOs:524-527,
SEQ ID NOs:529-534, SEQ ID NO:541, and SEQ ID NOs:544-545, where
the tissue has a difference in the level of oil as compared to the
corresponding level in tissue of a control plant that does not
comprise the nucleic acid.
[0021] The sequence identity can be 85 percent or greater, 90
percent or greater, or 95 percent or greater. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:81. The nucleotide sequence can encode a
polypeptide comprising an amino acid sequence corresponding to SEQ
ID NO:94. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:111.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:136. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:152. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:159. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:171.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:176. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:178. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:193. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:332.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:342. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:344. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:346. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:359.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:374. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:398. The difference can be an increase
in the level of oil. The exogenous nucleic acid can be operably
linked to a regulatory region. The regulatory region can be a
tissue-preferential regulatory region. The tissue-preferential
regulatory region can be a promoter. The regulatory region can be a
broadly expressing promoter. The plant tissue can be
dicotyledonous. The plant tissue can be a member of the genus
Anacardium, Arachis, Azadirachta, Brassica, Cannabis, Carthamus,
Corylus, Crambe, Cucurbita, Glycine, Gossypium, Helianthus,
Jatropha, Juglans, Linum, Olea, Papaver, Persea, Prunus, Ricinus,
Sesamum, Simmondsia, or Vitis. The plant tissue can be
monocotyledonous. The plant tissue can be a member of the genus
Cocos, Elaeis, Oryza, or Zea. The tissue can be seed tissue.
[0022] A plant cell is also provided. The plant cell comprises an
exogenous nucleic acid comprising a nucleotide sequence encoding a
polypeptide having 80 percent or greater sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs:81-92, SEQ ID NO:94, SEQ ID NOs:96-107, SEQ ID NO:109, SEQ ID
NOs:111-116, SEQ ID NOs:118-134, SEQ ID NOs:136-140, SEQ ID NO:142,
SEQ ID NO:144, SEQ ID NOs:146-147, SEQ ID NOs:149-150, SEQ ID
NO:152, SEQ ID NO:154, SEQ ID NOs:156-157, SEQ ID NOs:159-169, SEQ
ID NOs:171-172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NOs:178-179,
SEQ ID NOs:181-182, SEQ ID NOs:184-191, SEQ ID NOs:193-196, SEQ ID
NOs:198-217, SEQ ID NOs:219-220, SEQ ID NOs:222-229, SEQ ID
NOs:231-238, SEQ ID NO:240, SEQ ID NOs:242-250, SEQ ID NOs:252-330,
SEQ ID NOs:332-333, SEQ ID NO:335, SEQ ID NOs:337-340, SEQ ID
NO:342, SEQ ID NO:344, SEQ ID NO:346-348, SEQ ID NOs:350-357, SEQ
ID NOs:359-366, SEQ ID NO:368, SEQ ID NOs:370-372, SEQ ID
NOs:374-376, SEQ ID NOs:378-379, SEQ ID NOs:381-396, SEQ ID NO:398,
SEQ ID NOs:502-545, and the consensus sequences set forth in FIGS.
1-13, where a tissue of a plant produced from the plant cell has a
difference in the level of oil as compared to the corresponding
level in tissue of a control plant that does not comprise the
nucleic acid.
[0023] In another aspect, a plant cell is provided. The plant cell
comprises an exogenous nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ
ID NO:91, SEQ ID NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ
ID NO:109, SEQ ID NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID
NOs:120-121, SEQ ID NOs:125-132, SEQ ID NO:134, SEQ ID NO:136, SEQ
ID NOs:139-140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NOs:146-147,
SEQ ID NO:149, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NOs:156-157,
SEQ ID NO:159, SEQ ID NOs:162-168, SEQ ID NOs:171-172, SEQ ID
NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:184,
SEQ ID NOs:193-194, SEQ ID NO:198, SEQ ID NO:207, SEQ ID
NOs:215-217, SEQ ID NOs:219-220, SEQ ID NOs:222-223, SEQ ID
NOs:231-232, SEQ ID NO:235, SEQ ID NO:238, SEQ ID NO:240, SEQ ID
NO:242, SEQ ID NOs:244-246, SEQ ID NO:250, SEQ ID NO:252, SEQ ID
NO:254, SEQ ID NOs:257-258, SEQ ID NO:261, SEQ ID NOs:265-267, SEQ
ID NO:270, SEQ ID NO:279, SEQ ID NOs:287-293, SEQ ID NOs:300-301,
SEQ ID NO:304, SEQ ID NOs:307-309, SEQ ID NOs:311-313, SEQ ID
NOs:317-318, SEQ ID NO:320, SEQ ID NOs:323-324, SEQ ID NO:327, SEQ
ID NO:332, SEQ ID NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID
NO:344, SEQ ID NOs:346-348, SEQ ID NOs:350-351, SEQ ID NOs:355-357,
SEQ ID NOs:359-361, SEQ ID NOs:363-366, SEQ ID NO:368, SEQ ID
NOs:370-372, SEQ ID NO:374, SEQ ID NO:378, SEQ ID NO:381, SEQ ID
NO:398, SEQ ID NOs:502-505, SEQ ID NOs:507-519, SEQ ID NO:521, SEQ
ID NOs:524-527, SEQ ID NOs:529-534, SEQ ID NO:541, SEQ ID
NOs:544-545, and the consensus sequences set forth in FIGS. 1-13,
where a tissue of a plant produced from the plant cell has a
difference in the level of oil as compared to the corresponding
level in tissue of a control plant that does not comprise the
nucleic acid.
[0024] In another aspect, a plant cell is provided. The plant cell
comprises an exogenous nucleic acid comprising a nucleotide
sequence encoding a polypeptide having 80 percent or greater
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs:81-82, SEQ ID NOs:84-85, SEQ ID NO:87, SEQ
ID NO:91, SEQ ID NO:94, SEQ ID NOs:96-101, SEQ ID NOs:103-105, SEQ
ID NO:109, SEQ ID NO:111, SEQ ID NOs:113-115, SEQ ID NO:118, SEQ ID
NOs:120-121, SEQ ID NOs:125-132, SEQ ID NO:134, SEQ ID NO:136, SEQ
ID NOs:139-140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NOs:146-147,
SEQ ID NO:149, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NOs:156-157,
SEQ ID NO:159, SEQ ID NOs:162-168, SEQ ID NOs:171-172, SEQ ID
NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:184,
SEQ ID NOs:193-194, SEQ ID NO:198, SEQ ID NO:207, SEQ ID
NOs:215-217, SEQ ID NOs:219-220, SEQ ID NOs:222-223, SEQ ID
NOs:231-232, SEQ ID NO:235, SEQ ID NO:238, SEQ ID NO:240, SEQ ID
NO:242, SEQ ID NOs:244-246, SEQ ID NO:250, SEQ ID NO:252, SEQ ID
NO:254, SEQ ID NOs:257-258, SEQ ID NO:261, SEQ ID NOs:265-267, SEQ
ID NO:270, SEQ ID NO:279, SEQ ID NOs:287-293, SEQ ID NOs:300-301,
SEQ ID NO:304, SEQ ID NOs:307-309, SEQ ID NOs:311-313, SEQ ID
NOs:317-318, SEQ ID NO:320, SEQ ID NOs:323-324, SEQ ID NO:327, SEQ
ID NO:332, SEQ ID NO:335, SEQ ID NOs:337-340, SEQ ID NO:342, SEQ ID
NO:344, SEQ ID NOs:346-348, SEQ ID NOs:350-351, SEQ ID NOs:355-357,
SEQ ID NOs:359-361, SEQ ID NOs:363-366, SEQ ID NO:368, SEQ ID
NOs:370-372, SEQ ID NO:374, SEQ ID NO:378, SEQ ID NO:381, SEQ ID
NO:398, SEQ ID NOs:502-505, SEQ ID NOs:507-519, SEQ ID NO:521, SEQ
ID NOs:524-527, SEQ ID NOs:529-534, SEQ ID NO:541, and SEQ ID
NOs:544-545, where a tissue of a plant produced from the plant cell
has a difference in the level of oil as compared to the
corresponding level in tissue of a control plant that does not
comprise the nucleic acid.
[0025] The sequence identity can be 85 percent or greater, 90
percent or greater, or 95 percent or greater. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:81. The nucleotide sequence can encode a
polypeptide comprising an amino acid sequence corresponding to SEQ
ID NO:94. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:111.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:136. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:152. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:159. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:171.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:176. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:178. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:193. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:332.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:342. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:344. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to
SEQ ID NO:346. The nucleotide sequence can encode a polypeptide
comprising an amino acid sequence corresponding to SEQ ID NO:359.
The nucleotide sequence can encode a polypeptide comprising an
amino acid sequence corresponding to SEQ ID NO:374. The nucleotide
sequence can encode a polypeptide comprising an amino acid sequence
corresponding to SEQ ID NO:398. The nucleotide sequence can encode
a polypeptide comprising an amino acid sequence corresponding to a
consensus sequence set forth in FIG. 1, FIG. 2, FIG. 3, FIG. 4,
FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12,
or FIG. 13. The difference can be an increase in the level of oil.
The exogenous nucleic acid can be operably linked to a regulatory
region. The regulatory region can be a tissue-preferential
regulatory region. The tissue-preferential regulatory region can be
a promoter. The regulatory region can be a broadly expressing
promoter. The plant can be a dicot. The plant can be a member of
the genus Anacardium, Arachis, Azadirachta, Brassica, Cannabis,
Carthamus, Corylus, Crambe, Cucurbita, Glycine, Gossypium,
Helianthus, Jatropha, Juglans, Linum, Olea, Papaver, Persea,
Prunus, Ricinus, Sesamum, Simmondsia, or Vitis. The plant can be a
monocot. The plant can be a member of the genus Cocos, Elaeis,
Oryza, or Zea. The tissue can be seed tissue.
[0026] A transgenic plant is also provided. The transgenic plant
comprises any of the plant cells described above. Progeny of the
transgenic plant are also provided. The progeny have a difference
in the level of oil as compared to the level of oil in
corresponding control plants that do not comprise the exogenous
nucleic acid. Seed and vegetative tissue from the transgenic plant
are also provided. In addition, food products and feed products
comprising seed and/or vegetative tissue from the transgenic plant
are provided. Oil from the seed of the transgenic plant is
provided, as is a method of making oil. The method comprises
extracting oil from the seed of the transgenic plant.
[0027] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:95.
[0028] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:96.
[0029] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:108.
[0030] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:109.
[0031] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:117.
[0032] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:118.
[0033] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:141.
[0034] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:142.
[0035] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:143.
[0036] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:144.
[0037] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:145.
[0038] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:146.
[0039] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:148.
[0040] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:149.
[0041] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:153.
[0042] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:154.
[0043] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:155.
[0044] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:156.
[0045] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:173.
[0046] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:174.
[0047] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:180.
[0048] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:181.
[0049] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:183.
[0050] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:184.
[0051] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:197.
[0052] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:198.
[0053] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:218.
[0054] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:219.
[0055] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:221.
[0056] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:222.
[0057] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:230.
[0058] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:231.
[0059] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:239.
[0060] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:240.
[0061] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:241.
[0062] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:242.
[0063] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:251.
[0064] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:252.
[0065] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:270.
[0066] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:290.
[0067] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:292.
[0068] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:300.
[0069] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:308.
[0070] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:309.
[0071] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:318.
[0072] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:334.
[0073] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:335.
[0074] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:336.
[0075] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:337.
[0076] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:349.
[0077] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:350.
[0078] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:367.
[0079] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:368.
[0080] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:369.
[0081] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:370.
[0082] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:377.
[0083] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:378.
[0084] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:380.
[0085] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:381.
[0086] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:474.
[0087] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:475.
[0088] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:476.
[0089] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:477.
[0090] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:478.
[0091] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:479.
[0092] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:480.
[0093] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:481.
[0094] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:482.
[0095] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:483.
[0096] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:484.
[0097] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:485.
[0098] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:486.
[0099] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:487.
[0100] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:488.
[0101] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:489.
[0102] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:490.
[0103] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:491.
[0104] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:492.
[0105] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:493.
[0106] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:494.
[0107] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:495.
[0108] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:496.
[0109] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:497.
[0110] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:498.
[0111] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:499.
[0112] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:500.
[0113] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:501.
[0114] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:502.
[0115] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:503.
[0116] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:504.
[0117] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:505.
[0118] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:507.
[0119] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:509.
[0120] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:511.
[0121] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:513.
[0122] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:514.
[0123] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:515.
[0124] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:517.
[0125] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:519.
[0126] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:521.
[0127] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:525.
[0128] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:526.
[0129] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:527.
[0130] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:529.
[0131] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:531.
[0132] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:532.
[0133] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:533.
[0134] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:534.
[0135] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:541.
[0136] In another aspect, an isolated nucleic acid is provided. The
isolated nucleic acid comprises a nucleotide sequence encoding a
polypeptide having 80% or greater sequence identity to the amino
acid sequence set forth in SEQ ID NO:545.
[0137] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:546.
[0138] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:547.
[0139] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:549.
[0140] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:551.
[0141] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:553.
[0142] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:555.
[0143] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:556.
[0144] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:557.
[0145] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:559.
[0146] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:561.
[0147] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:562.
[0148] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:564.
[0149] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:565.
[0150] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:566.
[0151] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:567.
[0152] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:569.
[0153] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:570.
[0154] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:571.
[0155] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:572.
[0156] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:573.
[0157] In another aspect, an isolated nucleic acid molecule is
provided. The isolated nucleic acid molecule comprises a nucleotide
sequence having 95% or greater sequence identity to the nucleotide
sequence set forth in SEQ ID NO:575.
[0158] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0159] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0160] FIG. 1 is an alignment of Ceres Clone 41573 (SEQ ID NO:81)
with homologous and/or orthologous amino acid sequences Ceres
Clone:1560908 (SEQ ID NO:82), gi|2739168 (SEQ ID NO:83), Ceres
Clone:1314177 (SEQ ID NO:85), Ceres Clone 1371577 (SEQ ID NO:87),
and gi|50920801 (SEQ ID NO:89). The consensus sequence determined
by the alignment is set forth.
[0161] FIG. 2 is an alignment of Ceres Clone 25429 (SEQ ID NO:94)
with homologous and/or orthologous amino acid sequences Ceres
Annot:1488311 (SEQ ID NO:96), Ceres Clone:953928 (SEQ ID NO:97),
Ceres Clone:524682 (SEQ ID NO:98), Ceres Clone:1609735 (SEQ ID
NO:99), gi|42565379 (SEQ ID NO:102), Ceres Clone:426736 (SEQ ID
NO:103), and gi|24473796 (SEQ ID NO:107). The consensus sequence
determined by the alignment is set forth.
[0162] FIG. 3 is an alignment of Ceres Clone 5750 (SEQ ID NO:111)
with homologous and/or orthologous amino acid sequences gi|42362268
(SEQ ID NO:112), gi|27435806 (SEQ ID NO:116), gi|45935118 (SEQ ID
NO:119), Ceres Clone:1017141 (SEQ ID NO:120), Ceres Clone:1448636
(SEQ ID NO:121), gi|50919707 (SEQ ID NO:122), Ceres Clone:947192
(SEQ ID NO:127), and gi|55978016 (SEQ ID NO:133). The consensus
sequence determined by the alignment is set forth.
[0163] FIG. 4 is an alignment of Ceres Clone 218626 (SEQ ID NO:136)
with homologous and/or orthologous amino acid sequences gi|30409136
(SEQ ID NO:137), Ceres Clone:1571117 (SEQ ID NO:139), and Ceres
Annot:1440705 (SEQ ID NO:142). The consensus sequence determined by
the alignment is set forth.
[0164] FIG. 5 is an alignment of Ceres Clone 121021 (SEQ ID NO:152)
with homologous and/or orthologous amino acid sequences Ceres
Annot:1501628 (SEQ ID NO:154) and Ceres Clone:1121512 (SEQ ID
NO:157).
[0165] FIG. 6 is an alignment of Ceres Clone 158765 (SEQ ID NO:159)
with homologous and/or orthologous amino acid sequences gi|5669656
(SEQ ID NO:161), Ceres Clone:754061 (SEQ ID NO:162), Ceres
Clone:537752 (SEQ ID NO:164), Ceres Clone:282892 (SEQ ID NO:166),
and gi|50925813 (SEQ ID NO:169).
[0166] FIG. 7 is an alignment of Ceres Clone 16403 (SEQ ID NO:171)
with homologous and/or orthologous amino acid sequences Ceres
Clone:611156 (SEQ ID NO:172) and Ceres Annot:1464944 (SEQ ID
NO:174).
[0167] FIG. 8 is an alignment of Ceres Clone 28635 (SEQ ID NO:178)
with homologous and/or orthologous amino acid sequences gi|2463569
(SEQ ID NO:179), Ceres Annot:1514021 (SEQ ID NO:181), gi|55710094
(SEQ ID NO:182), gi|75859951 (SEQ ID NO:185), gi|1449163 (SEQ ID
NO:186), gi|28208268 (SEQ ID NO:188), gi|41224629 (SEQ ID NO:189),
gi|27475614 (SEQ ID NO:190), and gi|38426486 (SEQ ID NO:191).
[0168] FIG. 9 is an alignment of Clone 35698 (SEQ ID NO:193) with
homologous and/or orthologous amino acid sequences Clone
1380019.cndot.T (SEQ ID NO:266), gi|441457.cndot.T (SEQ ID NO:268),
Ceres Annot:1483290.cndot.T (SEQ ID NO:270), gi|40287554.cndot.T
(SEQ ID NO:271), gi|28569265.cndot.T (SEQ ID NO:274),
gi|22597164.cndot.T (SEQ ID NO:278), Clone 617835.cndot.T (SEQ ID
NO:279), gi|5762457.cndot.T (SEQ ID NO:281), gi|77416935.cndot.T
(SEQ ID NO:284), gi|28569267.cndot.T (SEQ ID NO:285),
gi|50906823.cndot.T (SEQ ID NO:295), gi|30025160.cndot.T (SEQ ID
NO:315), gi|54402104.cndot.T (SEQ ID NO:321), gi|52851174.cndot.T
(SEQ ID NO:326), and gi|4100646.cndot.T (SEQ ID NO:330).
[0169] FIG. 10 is an alignment of Ceres Clone 36412 (SEQ ID NO:332)
with homologous and/or orthologous amino acid sequences Ceres
Annot:1467033 (SEQ ID NO:335) and Ceres Clone:1641329 (SEQ ID
NO:338).
[0170] FIG. 11 is an alignment of Ceres Clone 4829 (SEQ ID NO:346)
with homologous and/or orthologous amino acid sequences Ceres
Annot:1485102 (SEQ ID NO:350), Ceres Clone:1646533 (SEQ ID NO:351),
gi|29371519 (SEQ ID NO:352), gi|45935148 (SEQ ID NO:354), Ceres
Clone:359934 (SEQ ID NO:355), and Ceres Clone:839270 (SEQ ID
NO:357).
[0171] FIG. 12 is an alignment of Ceres Clone 5426 (SEQ ID NO:359)
with homologous and/or orthologous amino acid sequences Ceres
Clone:1123542 (SEQ ID NO:360), Ceres Annot:1499194 (SEQ ID NO:368),
and Ceres Clone:557065 (SEQ ID NO:371).
[0172] FIG. 13 is an alignment of Ceres Clone 7894 (SEQ ID NO:374)
with homologous and/or orthologous amino acid sequences gi|18091781
(SEQ ID NO:375), gi|468562 (SEQ ID NO:376), Ceres Annot:1479767
(SEQ ID NO:378), gi|7649151 (SEQ ID NO:379), gi|33620334 (SEQ ID
NO:382), gi|439294 (SEQ ID NO:383), gi|5230818 (SEQ ID NO:385),
gi|17447420 (SEQ ID NO:386), gi|1935019 (SEQ ID NO:387),
gi|51863031 (SEQ ID NO:388), gi|575351 (SEQ ID NO:389), gi|68161544
(SEQ ID NO:390), gi|21319 (SEQ ID NO:392), gi|5823000 (SEQ ID
NO:393), gi|6120115 (SEQ ID NO:395), and gi|415988 (SEQ ID
NO:396).
DETAILED DESCRIPTION
[0173] The invention features methods and materials related to
modulating (e.g., increasing or decreasing) oil levels in plants.
In some embodiments, the plants may also have modulated levels of
protein. The methods can include transforming a plant cell with a
nucleic acid encoding an oil-modulating polypeptide, wherein
expression of the polypeptide results in a modulated level of oil.
Plant cells produced using such methods can be grown to produce
plants having an increased or decreased oil content. Seeds from
such plants may be used to produce, for example, foodstuffs and
animal feed having an increased oil content. Producing oil from
seeds having an increased oil content can allow manufacturers to
increase oil yields.
Polypeptides
[0174] The term "polypeptide" as used herein refers to a compound
of two or more subunit amino acids, amino acid analogs, or other
peptidomimetics, regardless of post-translational modification,
e.g., phosphorylation or glycosylation. The subunits may be linked
by peptide bonds or other bonds such as, for example, ester or
ether bonds. The term "amino acid" refers to natural and/or
unnatural or synthetic amino acids, including D/L optical isomers.
Full-length proteins, analogs, mutants, and fragments thereof are
encompassed by this definition.
[0175] Polypeptides described herein include oil-modulating
polypeptides. Oil-modulating polypeptides can be effective to
modulate oil levels when expressed in a plant or plant cell.
Modulation of the level of oil can be either an increase or a
decrease in the level of oil relative to the corresponding level in
a control plant.
[0176] An oil-modulating polypeptide can be an enzyme, such as an
aldose 1-epimerase, a squalene/phytoene synthase, a
ubiquitin-conjugating enzyme, or a cytochrome p450 enzyme. An
oil-modulating polypeptide can also be a ribosomal polypeptide,
such as a 60S acidic ribosomal protein or a ribosomal protein L28e.
An oil-modulating polypeptide can also be a regulatory polypeptide,
such as a cyclin-dependent kinase regulatory subunit or a PsbP
protein subunit of photosystem II (PSII). An oil-modulating
polypeptide can also be a transporter polypeptide, such as a
tryptophan/tyrosine permease or a major facilitator superfamily
(MFS) transporter. An oil-modulating polypeptide can also be a
transcription factor polypeptide, such as an AP2 domain-containing
transcription factor polypeptide.
[0177] An oil-modulating polypeptide can be an enzyme involved in
carbohydrate metabolism, such as an aldose 1-epimerase family
polypeptide. Aldose 1-epimerase catalyzes the interconversion of
the alpha- and beta-anomers of hexose sugars such as glucose and
galactose. SEQ ID NO:81 sets forth the amino acid sequence of an
Arabidopsis clone, identified herein as Ceres Clone 41573 (SEQ ID
NO:80), that is predicted to encode an aldose 1-epimerase family
polypeptide. An oil-modulating polypeptide can comprise the amino
acid sequence set forth in SEQ ID NO:81. Alternatively, an
oil-modulating polypeptide can be a homolog, ortholog, or variant
of the polypeptide having the amino acid sequence set forth in SEQ
ID NO:81. For example, an oil-modulating polypeptide can have an
amino acid sequence with at least 45% sequence identity, e.g., 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%
sequence identity, to the amino acid sequence set forth in SEQ ID
NO:81.
[0178] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:81 are provided in FIG. 1, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:81, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 1 provides the
amino acid sequences of Ceres Clone 41573 (SEQ ID NO:81), Ceres
Clone:1560908 (SEQ ID NO:82), gi|2739168 (SEQ ID NO:83), Ceres
Clone:1314177 (SEQ ID NO:85), Ceres Clone 1371577 (SEQ ID NO:87),
and gi|50920801 (SEQ ID NO:89). Other homologs and/or orthologs
include Ceres CLONE ID no. 399052 (SEQ ID NO:84), Public GI no.
15824567 (SEQ ID NO:86), Public GI no. 15824565 (SEQ ID NO:88),
Public GI no. 50909807 (SEQ ID NO:90), Ceres CLONE ID no. 639223
(SEQ ID NO:91), Public GI no. 37531218 (SEQ ID NO:92), and Ceres
Cone:1476735 (SEQ ID NO:502).
[0179] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84,
SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID
NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:502, or
the consensus sequence set forth in FIG. 1.
[0180] An oil-modulating polypeptide can be a ribosomal
polypeptide, such as a 60S acidic ribosomal polypeptide. SEQ ID
NO:94 sets forth the amino acid sequence of an Arabidopsis clone,
identified herein as Ceres Clone 25429 (SEQ ID NO:93), that is
predicted to encode a 60S acidic ribosomal polypeptide. An
oil-modulating polypeptide can comprise the amino acid sequence set
forth in SEQ ID NO:94. Alternatively, an oil-modulating polypeptide
can be a homolog, ortholog, or variant of the polypeptide having
the amino acid sequence set forth in SEQ ID NO:94. For example, an
oil-modulating polypeptide can have an amino acid sequence with at
least 60% sequence identity, e.g., 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid
sequence set forth in SEQ ID NO:94.
[0181] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:94 are provided in FIG. 2, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:94, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 2 provides the
amino acid sequences of Ceres Clone 25429 (SEQ ID NO:94), Ceres
Annot:1488311 (SEQ ID NO:96), Ceres Clone:953928 (SEQ ID NO:97),
Ceres Clone:524682 (SEQ ID NO:98), Ceres Clone:1609735 (SEQ ID
NO:99), gi|42565379 (SEQ ID NO:102), Ceres Clone:426736 (SEQ ID
NO:103), and gi|24473796 (SEQ ID NO:107). Other homologs and/or
orthologs include Ceres CLONE ID no. 949174 (SEQ ID NO:100), Ceres
CLONE ID no. 1299820 (SEQ ID NO:101), Ceres CLONE ID no. 1094375
(SEQ ID NO:104), Ceres CLONE ID no. 691062 (SEQ ID NO:105), Public
GI no. 47026878 (SEQ ID NO:106), Ceres Annot:1465437_PRT (SEQ ID
NO:109), Ceres Clone:1798334 (SEQ ID NO:503), Ceres Clone:1886478
(SEQ ID NO:504), Ceres Clone:1727128 (SEQ ID NO:505), and gi|730583
(SEQ ID NO:506).
[0182] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98,
SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107,
SEQ ID NO:109, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ ID
NO:506, or the consensus sequence set forth in FIG. 2.
[0183] An oil-modulating polypeptide can be a cyclin-dependent
kinase regulatory subunit. In eukaryotes, cyclin-dependent protein
kinases interact with cyclins to regulate cell cycle progression.
The proteins bind to a regulatory subunit, cyclin-dependent kinase
regulatory subunit (CKS), which is essential for their function.
SEQ ID NO:111 sets forth the amino acid sequence of an Arabidopsis
clone, identified herein as Ceres Clone 5750 (SEQ ID NO:110), that
is predicted to encode a cyclin-dependent kinase regulatory
subunit. An oil-modulating polypeptide can comprise the amino acid
sequence set forth in SEQ ID NO:111. Alternatively, an
oil-modulating polypeptide can be a homolog, ortholog, or variant
of the polypeptide having the amino acid sequence set forth in SEQ
ID NO:111. For example, an oil-modulating polypeptide can have an
amino acid sequence with at least 75% sequence identity, e.g., 75%,
80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
amino acid sequence set forth in SEQ ID NO:111.
[0184] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:111 are provided in FIG. 3, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:111, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 3 provides the
amino acid sequences of Ceres Clone 5750 (SEQ ID NO:111),
gi|42362268 (SEQ ID NO:112), gi|27435806 (SEQ ID NO:116),
gi|45935118 (SEQ ID NO:119), Ceres Clone:1017141 (SEQ ID NO:120),
Ceres Clone:1448636 (SEQ ID NO:121), gi|50919707 (SEQ ID NO:122),
Ceres Clone:947192 (SEQ ID NO:127), and gi|55978016 (SEQ ID
NO:133). Other homologs and/or orthologs include Ceres CLONE ID no.
709027 (SEQ ID NO:113), Ceres CLONE ID no. 853298 (SEQ ID NO:114),
Ceres CLONE ID no. 1417425 (SEQ ID NO:115), Ceres Annot:1481954
(SEQ ID NO:118), Public GI no. 40641585 (SEQ ID NO:123), Public GI
no. 38566522 (SEQ ID NO:124), Ceres CLONE ID no. 1338131 (SEQ ID
NO:125), Ceres CLONE ID no. 300692 (SEQ ID NO:126), Ceres CLONE ID
no. 1465004 (SEQ ID NO:128), Ceres CLONE ID no. 1122958 (SEQ ID
NO:129), Ceres CLONE ID no. 944737 (SEQ ID NO:130), Ceres CLONE ID
no. 217797 (SEQ ID NO:131), Ceres CLONE ID no. 520185 (SEQ ID
NO:132), Ceres CLONE ID no. 1436585 (SEQ ID NO:134), Ceres
Clone:1777369 (SEQ ID NO:507), Ceres Clone:1744578 (SEQ ID NO:508),
Ceres Clone: 100008703 (SEQ ID NO:509), and Ceres Clone:1723582
(SEQ ID NO:510).
[0185] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:112, SEQ ID NO:113, SEQ ID
NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:119,
SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID
NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,
SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:133, SEQ ID NO:134, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509,
SEQ ID NO:510, or the consensus sequence set forth in FIG. 3.
[0186] An oil-modulating polypeptide can be a tryptophan/tyrosine
permease family polypeptide. Amino acid permeases are integral
membrane proteins involved in the transport of amino acids into the
cell. SEQ ID NO:136 sets forth the amino acid sequence of a Zea
mays clone, identified herein as Ceres Clone 218626 (SEQ ID
NO:135), that is predicted to encode a tryptophan/tyrosine permease
family polypeptide. The tryptophan/tyrosine permease family of
proteins is characterized by, inter alia, the presence of several
membrane-spanning domains. Members of the tryptophan/tyrosine
permease family sometimes have homology to other permeases and
transporters. An oil-modulating polypeptide can comprise the amino
acid sequence set forth in SEQ ID NO:136. Alternatively, an
oil-modulating polypeptide can be a homolog, ortholog, or variant
of the polypeptide having the amino acid sequence set forth in SEQ
ID NO:136. For example, an oil-modulating polypeptide can have an
amino acid sequence with at least 70% sequence identity, e.g., 70%,
75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
amino acid sequence set forth in SEQ ID NO:136.
[0187] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:136 are provided in FIG. 4, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:136, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 4 provides the
amino acid sequences of Ceres Clone 218626 (SEQ ID NO:136),
gi|30409136 (SEQ ID NO:137), Ceres Clone:1571117 (SEQ ID NO:139),
and Ceres Annot:1440705 (SEQ ID NO:142). Other homologs and/or
orthologs include Public GI no. 50940751 (SEQ ID NO:138), Ceres
CLONE ID no. 424395 (SEQ ID NO:140), Ceres Annot:1493584 (SEQ ID
NO:144), Ceres Annot:1463076 (SEQ ID NO:146), Ceres CLONE ID no.
1002421 (SEQ ID NO:147), Ceres Annot:1516369 (SEQ ID NO:149),
Public GI no. 30693666 (SEQ ID NO:150), and Ceres Clone:1796001
(SEQ ID NO:511).
[0188] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:137, SEQ ID NO:138, SEQ ID
NO:139, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146,
SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:511, or the
consensus sequence set forth in FIG. 4.
[0189] An oil-modulating polypeptide can be a polypeptide that does
not have homology to an existing protein family based on Pfam
analysis. SEQ ID NO:152 sets forth the amino acid sequence of an
Arabidopsis clone, identified herein as Ceres Clone 121021 (SEQ ID
NO:151), that is predicted to encode a polypeptide that does not
have homology to an existing protein family based on Pfam analysis.
An oil-modulating polypeptide can comprise the amino acid sequence
set forth in SEQ ID NO:152. Alternatively, an oil-modulating
polypeptide can be a homolog having at least 45% sequence identity,
e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% sequence identity, to the amino acid sequence set forth
in SEQ ID NO:152.
[0190] Amino acid sequences of homologs of the polypeptide having
the amino acid sequence set forth in SEQ ID NO:152 are provided in
FIG. 5, along with a consensus sequence. A consensus amino acid
sequence for such homologs was determined by aligning amino acid
sequences, e.g., amino acid sequences related to SEQ ID NO:152,
from a variety of species and determining the most common amino
acid or type of amino acid at each position. For example, the
alignment in FIG. 5 provides the amino acid sequences of Ceres
Clone 121021 (SEQ ID NO:152), Ceres Annot:1501628 (SEQ ID NO:154)
and Ceres Clone:1121512 (SEQ ID NO:157). Other homologs include
Ceres Annot:1519046 (SEQ ID NO:156).
[0191] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:154, SEQ ID NO:156, SEQ ID
NO:157, or the consensus sequence set forth in FIG. 5.
[0192] SEQ ID NO:159 sets forth the amino acid sequence of another
Arabidopsis clone, identified herein as Ceres Clone 158765 (SEQ ID
NO:158), that is predicted to encode a polypeptide that does not
have homology to an existing protein family based on Pfam analysis.
An oil-modulating polypeptide can comprise the amino acid sequence
set forth in SEQ ID NO:159. Alternatively, an oil-modulating
polypeptide can be a homolog having at least 45% sequence identity,
e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% sequence identity, to the amino acid sequence set forth
in SEQ ID NO:159.
[0193] Amino acid sequences of homologs of the polypeptide having
the amino acid sequence set forth in SEQ ID NO:159 are provided in
FIG. 6, along with a consensus sequence. A consensus amino acid
sequence for such homologs was determined by aligning amino acid
sequences, e.g., amino acid sequences related to SEQ ID NO:159,
from a variety of species and determining the most common amino
acid or type of amino acid at each position. For example, the
alignment in FIG. 6 provides the amino acid sequences of Ceres
Clone 158765 (SEQ ID NO:159), gi|5669656 (SEQ ID NO:161), Ceres
Clone:754061 (SEQ ID NO:162), Ceres Clone:537752 (SEQ ID NO:164),
Ceres Clone:282892 (SEQ ID NO:166), and gi|50925813 (SEQ ID
NO:169). Other homologs include Public GI no. 32562996 (SEQ ID
NO:160), Ceres CLONE ID no. 1329861 (SEQ ID NO:163), Ceres CLONE ID
no. 1322549 (SEQ ID NO:165), Ceres CLONE ID no. 284046 (SEQ ID
NO:167), Ceres CLONE ID no. 1388825 (SEQ ID NO:168), and Ceres
Clone:1839717 (SEQ ID NO:545).
[0194] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:160, SEQ ID NO:161, SEQ ID
NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166,
SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:545, or the
consensus sequence set forth in FIG. 6. An oil-modulating
polypeptide can be a PsbP polypeptide. A PsbP polypeptide is a
subunit of photosystem II (PSII) that is reported to be essential
for the regulation and stabilization of PSII in higher plants. SEQ
ID NO:171 sets forth the amino acid sequence of an Arabidopsis
clone, identified herein as Ceres Clone 16403 (SEQ ID NO:170), that
is predicted to encode a PsbP polypeptide. An oil-modulating
polypeptide can comprise the amino acid sequence set forth in SEQ
ID NO:171. Alternatively, an oil-modulating polypeptide can be a
homolog, ortholog, or variant of the polypeptide having the amino
acid sequence set forth in SEQ ID NO:171. For example, an
oil-modulating polypeptide can have an amino acid sequence with at
least 50% sequence identity, e.g., 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
amino acid sequence set forth in SEQ ID NO:171.
[0195] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:171 are provided in FIG. 7, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:171, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 7 provides the
amino acid sequences of Ceres Clone 16403 (SEQ ID NO:171), Ceres
Clone:611156 (SEQ ID NO:172), Ceres Annot:1464944 (SEQ ID NO:174),
Ceres Clone:1728680 (SEQ ID NO:530), Ceres Clone:1807796 (SEQ ID
NO:531), Ceres Clone:1771837 (SEQ ID NO:532), and Ceres
Clone:1773482 (SEQ ID NO:533).
[0196] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:172, SEQ ID NO:174, SEQ ID
NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, or the
consensus sequence set forth in FIG. 7.
[0197] An oil-modulating polypeptide can be a transcription factor
that contains an AP2 (APETALA2) DNA-binding domain. AP2 is one of
the prototypic members of a family of transcription factors unique
to plants, whose distinguishing characteristic is that they contain
the so-called AP2 DNA-binding domain. SEQ ID NO:176 sets forth the
amino acid sequence of an Arabidopsis clone, identified herein as
Ceres Clone 19244 (SEQ ID NO:175), that is predicted to encode a
transcription factor containing an AP2 DNA-binding domain. An
oil-modulating polypeptide can comprise the amino acid sequence set
forth in SEQ ID NO:176. Alternatively, an oil-modulating
polypeptide can be a homolog, ortholog, or variant of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:176. For example, an oil-modulating polypeptide can have an
amino acid sequence with at least 40% sequence identity, e.g., 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or
99% sequence identity, to the amino acid sequence set forth in SEQ
ID NO:176.
[0198] An oil-modulating polypeptide can be a squalene/phytoene
synthase. Squalene synthase catalyzes the conversion of two
molecules of farnesyl diphosphate into squalene, which is the first
committed step in the cholesterol biosynthetic pathway. Phytoene
synthase catalyzes the conversion of two molecules of
geranylgeranyl diphosphate into phytoene, which is the second step
in the biosynthesis of carotenoids from isopentenyl diphosphate.
SEQ ID NO:178 sets forth the amino acid sequence of an Arabidopsis
clone, identified herein as Ceres Clone 28635 (SEQ ID NO:177), that
is predicted to encode a squalene/phytoene synthase. An
oil-modulating polypeptide can comprise the amino acid sequence set
forth in SEQ ID NO:178. Alternatively, an oil-modulating
polypeptide can be a homolog, ortholog, or variant of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:178. For example, an oil-modulating polypeptide can have an
amino acid sequence with at least 70% sequence identity, e.g., 70%,
75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
amino acid sequence set forth in SEQ ID NO:178.
[0199] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:178 are provided in FIG. 8, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:178, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 8 provides the
amino acid sequences of Ceres Clone 28635 (SEQ ID NO:178),
gi|2463569 (SEQ ID NO:179), Ceres Annot:1514021 (SEQ ID NO:181),
gi|55710094 (SEQ ID NO:182), gi/75859951 (SEQ ID NO:185),
gi|1449163 (SEQ ID NO:186), gi|28208268 (SEQ ID NO:188),
gi|41224629 (SEQ ID NO:189), gi|27475614 (SEQ ID NO:190), and
gi|38426486 (SEQ ID NO:191). Other homologs and/or orthologs
include Ceres Annot:1503464 (SEQ ID NO:184) and Public GI no.
1449165 (SEQ ID NO:187), Ceres Clone:1920025 (SEQ ID NO:534),
gi|110293133 (SEQ ID NO:535), gi|5360655 (SEQ ID NO:536),
gi|4426953 (SEQ ID NO:537), gi|1552717 (SEQ ID NO:538), gi|66393825
(SEQ ID NO:539), gi|1706774 (SEQ ID NO:540), Ceres Clone:1749989
(SEQ ID NO:541), gi|115456049 (SEQ ID NO:542), gi|2463567 (SEQ ID
NO:543), and Ceres Clone:706088 (SEQ ID NO:544).
[0200] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:179, SEQ ID NO:181, SEQ ID
NO:182, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187,
SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID
NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538,
SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ ID NO:542, SEQ ID
NO:543, SEQ ID NO:544, or the consensus sequence set forth in FIG.
8.
[0201] An oil-modulating polypeptide can be an
ubiquitin-conjugating enzyme. An ubiquitin-conjugating enzyme (E2)
is one of at least three enzymes involved in ubiquitination. The E2
enzyme transfers a ubiquitin moiety directly to a substrate, or to
a ubiquitin ligase (E3). E2 enzymes are broadly grouped into four
classes: class I enzymes contain the catalytic core domain (UBC)
having an active site cysteine, class II enzymes possess a UBC and
a C-terminal extension, class III enzymes possess a UBC and an
N-terminal extension, and class IV enzymes possess a UBC and both
N- and C-terminal extensions. These extensions appear to be
important for some subfamily function, including E2 localization
and protein-protein interactions. In addition, there are proteins
with an E2-like fold that are devoid of catalytic activity, but
which appear to assist in poly-ubiquitin chain formation. SEQ ID
NO:193 sets forth the amino acid sequence of an Arabidopsis clone,
identified herein as Ceres Clone 35698 (SEQ ID NO:192), that is
predicted to encode a ubiquitin-conjugating enzyme. An
oil-modulating polypeptide can comprise the amino acid sequence set
forth in SEQ ID NO:193. Alternatively, an oil-modulating
polypeptide can be a homolog, ortholog, or variant of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:193. For example, an oil-modulating polypeptide can have an
amino acid sequence with at least 75% sequence identity, e.g., 75%,
80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
amino acid sequence set forth in SEQ ID NO:193.
[0202] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:193 are provided in FIG. 9, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:193, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 9 provides the
amino acid sequences of Ceres Clone 35698 (SEQ ID NO:193), Clone
1380019.cndot.T (SEQ ID NO:266), gi|441457.cndot.T (SEQ ID NO:268),
Ceres Annot:1483290.cndot.T (SEQ ID NO:270), gi|40287554.cndot.T
(SEQ ID NO:271), gi|28569265.cndot.T (SEQ ID NO:274),
gi|22597164.cndot.T (SEQ ID NO:278), Clone 617835.cndot.T (SEQ ID
NO:279), gi|5762457.cndot.T (SEQ ID NO:281), gi|77416935.cndot.T
(SEQ ID NO:284), gi|28569267.cndot.T (SEQ ID NO:285),
gi|50906823.cndot.T (SEQ ID NO:295), gi|30025160.cndot.T (SEQ ID
NO:315), gi|54402104.cndot.T (SEQ ID NO:321), gi|52851174.cndot.T
(SEQ ID NO:326), and gi|4100646.cndot.T (SEQ ID NO:330). Other
homologs and/or orthologs include Ceres CLONE ID no. 1346445 (SEQ
ID NO:194), Public GI no. 441457 (SEQ ID NO:195), Public GI no.
19347859 (SEQ ID NO:196), Ceres Annot:1483290 (SEQ ID NO:198),
Public GI no. 40287554 (SEQ ID NO:199), Public GI no. 21553796 (SEQ
ID NO:200), Public GI no. 28569271 (SEQ ID NO:201), Public GI no.
28569265 (SEQ ID NO:202), Public GI no. 66354420 (SEQ ID NO:203),
Public GI no. 21280893 (SEQ ID NO:204), Public GI no. 21554343 (SEQ
ID NO:205), Public GI no. 22597164 (SEQ ID NO:206), Ceres CLONE ID
no. 617835 (SEQ ID NO:207), Public GI no. 30693871 (SEQ ID NO:208),
Public GI no. 5762457 (SEQ ID NO:209), Public GI no. 464981 (SEQ ID
NO:210), Public GI no. 456568 (SEQ ID NO:211), Public GI no.
77416935 (SEQ ID NO:212), Public GI no. 28569267 (SEQ ID NO:213),
Public GI no. 28569261 (SEQ ID NO:214), Ceres CLONE ID no. 39130
(SEQ ID NO:215), Ceres CLONE ID no. 16865 (SEQ ID NO:216), Ceres
CLONE ID no. 575067 (SEQ ID NO:217), Ceres Annot:1467392 (SEQ ID
NO:219), Ceres CLONE ID no. 25162 (SEQ ID NO:220), Ceres
Annot:1529647 (SEQ ID NO:222), Ceres CLONE ID no. 1405728 (SEQ ID
NO:223), Public GI no. 54288726 (SEQ ID NO:224), Public GI no.
50906823 (SEQ ID NO:225), Public GI no. 83306206 (SEQ ID NO:226),
Public GI no. 20152203 (SEQ ID NO:227), Public GI no. 40287568 (SEQ
ID NO:228), Public GI no. 50929483 (SEQ ID NO:229), Ceres
Annot:1450556 (SEQ ID NO:231), Ceres CLONE ID no. 1031152 (SEQ ID
NO:232), Public GI no. 52548244 (SEQ ID NO:233), Public GI no.
82621144 (SEQ ID NO:234), Ceres CLONE ID no. 878043 (SEQ ID
NO:235), Public GI no. 34909292 (SEQ ID NO:236), Public GI no.
2668744 (SEQ ID NO:237), Ceres CLONE ID no. 511132 (SEQ ID NO:238),
Ceres Annot:1533930 (SEQ ID NO:240), Ceres Annot:1495171 (SEQ ID
NO:242), Public GI no. 20086317 (SEQ ID NO:243), Ceres CLONE ID no.
10022 (SEQ ID NO:244), Ceres CLONE ID no. 12547 (SEQ ID NO:245),
Ceres CLONE ID no. 27679 (SEQ ID NO:246), Public GI no. 20259611
(SEQ ID NO:247), Public GI no. 30025160 (SEQ ID NO:248), Public GI
no. 50904839 (SEQ ID NO:249), Ceres CLONE ID no. 1063753 (SEQ ID
NO:250), Ceres Annot:1533218 (SEQ ID NO:252), Public GI no. 297880
(SEQ ID NO:253), Ceres CLONE ID no. 1357060 SEQ ID NO:254), Public
GI no. 54402104 (SEQ ID NO:255), Public GI no. 50725323 (SEQ ID
NO:256), Ceres CLONE ID no. 376667 (SEQ ID NO:257), Ceres CLONE ID
no. 256705 (SEQ ID NO:258), Public GI no. 20259629 (SEQ ID NO:259),
Public GI no. 52851174 (SEQ ID NO:260), Ceres CLONE ID no. 1061097
(SEQ ID NO:261), Public GI no. 1373001 (SEQ ID NO:262), Public GI
no. 66354468 (SEQ ID NO:263), Public GI no. 4100646 (SEQ ID
NO:264), Ceres CLONE ID no. 1380019 (SEQ ID NO:265), Ceres CLONE ID
no. 1346445_T (SEQ ID NO:267), Public GI no. 19347859_T (SEQ ID
NO:269), Public GI no. 21553796_T (SEQ ID NO:272), Public GI no.
28569271_T (SEQ ID NO:273), Public GI no. 66354420_T (SEQ ID
NO:275), Public GI no. 21280893_T (SEQ ID NO:276), Public GI no.
21554343_T (SEQ ID NO:277), Public GI no. 30693871_T (SEQ ID
NO:280), Public GI no. 464981_T (SEQ ID NO:282), Public GI no.
456568_T (SEQ ID NO:283), Public GI no. 28569261_T (SEQ ID NO:286),
Ceres CLONE ID no. 39130_T (SEQ ID NO:287), Ceres CLONE ID no.
16865_T (SEQ ID NO:288), Ceres CLONE ID no. 575067_T (SEQ ID
NO:289), Ceres Annot:1467392_T (SEQ ID NO:290), Ceres CLONE ID no.
25162_T (SEQ ID NO:291), Ceres Annot:1529647_T (SEQ ID NO:292),
Ceres CLONE ID no. 1405728_T (SEQ ID NO:293), Public GI no.
54288726_T (SEQ ID NO:294), Public GI no. 83306206_T (SEQ ID
NO:296), Public GI no. 20152203_T (SEQ ID NO:297), Public GI no.
40287568_T (SEQ ID NO:298), Public GI no. 50929483_T (SEQ ID
NO:299), Ceres Annot:1450556_T (SEQ ID NO:300), Ceres CLONE ID no.
1031152_T (SEQ ID NO:301), Public GI no. 52548244_T (SEQ ID
NO:302), Public GI no. 82621144_T (SEQ ID NO:303), Ceres CLONE ID
no. 878043_T (SEQ ID NO:304), Public GI no. 34909292_T (SEQ ID
NO:305), Public GI no. 2668744_T (SEQ ID NO:306), Ceres CLONE ID
no. 511132_T (SEQ ID NO:307), Ceres Annot:1533930_T (SEQ ID
NO:308), Ceres Annot:1495171_T (SEQ ID NO:309), Public GI no.
20086317_T (SEQ ID NO:310), Ceres CLONE ID no. 10022_T (SEQ ID
NO:311), Ceres CLONE ID no. 12547_T (SEQ ID NO:312), Ceres CLONE ID
no. 27679_T (SEQ ID NO:313), Public GI no. 20259611_T (SEQ ID
NO:314), Public GI no. 50904839_T (SEQ ID NO:316), Ceres CLONE ID
no. 1063753_T (SEQ ID NO:317), Ceres Annot:1533218_T (SEQ ID
NO:318), Public GI no. 297880_T (SEQ ID NO:319), Ceres CLONE ID no.
1357060_T (SEQ ID NO:320), Public GI no. 50725323_T (SEQ ID
NO:322), Ceres CLONE ID no. 376667_T (SEQ ID NO:323), Ceres CLONE
ID no. 256705_T (SEQ ID NO:324), Public GI no. 20259629_T (SEQ ID
NO:325), Ceres CLONE ID no. 1061097_T (SEQ ID NO:327), Public GI
no. 1373001_T (SEQ ID NO:328), and Public GI no. 66354468_T (SEQ ID
NO:329).
[0203] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:194, SEQ ID NO:195, SEQ ID
NO:196, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201,
SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210,
SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID
NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:220,
SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID
NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:231,
SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID
NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242,
SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID
NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:252,
SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID
NO:257, SEQ ID NO:258, SEQ ID NO:259, SEQ ID NO:260, SEQ ID NO:261,
SEQ ID NO:262, SEQ ID NO:263, SEQ ID NO:264, SEQ ID NO:265, SEQ ID
NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270,
SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID
NO:275, SEQ ID NO:276, SEQ ID NO:277, SEQ ID NO:278, SEQ ID NO:279,
SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, SEQ ID
NO:284, SEQ ID NO:285, SEQ ID NO:286, SEQ ID NO:287, SEQ ID NO:288,
SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:292, SEQ ID
NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297,
SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID
NO:302, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306,
SEQ ID NO:307, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID
NO:311, SEQ ID NO:312, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315,
SEQ ID NO:316, SEQ ID NO:317, SEQ ID NO:318, SEQ ID NO:319, SEQ ID
NO:320, SEQ ID NO:321, SEQ ID NO:322, SEQ ID NO:323, SEQ ID NO:324,
SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327, SEQ ID NO:328, SEQ ID
NO:329, SEQ ID NO:330, or the consensus sequence set forth in FIG.
9.
[0204] SEQ ID NO:332 sets forth the amino acid sequence of another
Arabidopsis clone, identified herein as Ceres Clone 36412 (SEQ ID
NO:331), that is predicted to encode a polypeptide that does not
have homology to an existing protein family based on Pfam analysis.
An oil-modulating polypeptide can comprise the amino acid sequence
set forth in SEQ ID NO:332. Alternatively, an oil-modulating
polypeptide can be a variant having at least 45% sequence identity,
e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% sequence identity, to the amino acid sequence set forth
in SEQ ID NO:332.
[0205] Amino acid sequences of homologs of the polypeptide having
the amino acid sequence set forth in SEQ ID NO:332 are provided in
FIG. 10, along with a consensus sequence. A consensus amino acid
sequence for such homologs was determined by aligning amino acid
sequences, e.g., amino acid sequences related to SEQ ID NO:332,
from a variety of species and determining the most common amino
acid or type of amino acid at each position. For example, the
alignment in FIG. 10 provides the amino acid sequences of Ceres
Clone 36412 (SEQ ID NO:332), Ceres Annot:1467033 (SEQ ID NO:335)
and Ceres Clone:1641329 (SEQ ID NO:338). Other homologs include
Public GI no. 3152583 (SEQ ID NO:333), Ceres Annot:1536919 (SEQ ID
NO:337), Ceres CLONE ID no. 1650419 (SEQ ID NO:339), and Ceres
CLONE ID no. 597699 (SEQ ID NO:340).
[0206] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:333, SEQ ID NO:335, SEQ ID
NO:337, SEQ ID NO:338, SEQ ID NO:339, SEQ ID NO:340, or the
consensus sequence set forth in FIG. 10.
[0207] SEQ ID NO:342 sets forth the amino acid sequence of another
Arabidopsis clone, identified herein as Ceres Clone 368 (SEQ ID
NO:341), that is predicted to encode a polypeptide that does not
have homology to an existing protein family based on Pfam analysis.
An oil-modulating polypeptide can comprise the amino acid sequence
set forth in SEQ ID NO:342. Alternatively, an oil-modulating
polypeptide can be a homolog having at least 40% sequence identity,
e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 98%, or 99% sequence identity, to the amino acid sequence set
forth in SEQ ID NO:342
[0208] An oil-modulating polypeptide can be a cytochrome p450
polypeptide. Cytochrome p450 enzymes are haem-thiolate polypeptides
involved in the oxidative modification of various compounds. SEQ ID
NO:344 sets forth the amino acid sequence of an Arabidopsis clone,
identified herein as Ceres Clone 41046 (SEQ ID NO:343), that is
predicted to encode a cytochrome p450 enzyme. An oil-modulating
polypeptide can comprise the amino acid sequence set forth in SEQ
ID NO:344. Alternatively, an oil-modulating polypeptide can be a
homolog, ortholog, or variant of the polypeptide having the amino
acid sequence set forth in SEQ ID NO:344. For example, an
oil-modulating polypeptide can have an amino acid sequence with at
least 40% sequence identity, e.g., 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,
to the amino acid sequence set forth in SEQ ID NO:344.
[0209] An oil-modulating polypeptide can have a DUF662 domain
characteristic of a family of hypothetical eukaryotic proteins. SEQ
ID NO:346 sets forth the amino acid sequence of an Arabidopsis
clone, identified herein as Ceres Clone 4829 (SEQ ID NO:345), that
is predicted to encode a eukaryotic polypeptide. An oil-modulating
polypeptide can comprise the amino acid sequence set forth in SEQ
ID NO:346. Alternatively, an oil-modulating polypeptide can be a
homolog, ortholog, or variant of the polypeptide having the amino
acid sequence set forth in SEQ ID NO:346. For example, an
oil-modulating polypeptide can have an amino acid sequence with at
least 50% sequence identity, e.g., 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the
amino acid sequence set forth in SEQ ID NO:346.
[0210] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:346 are provided in FIG. 11, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:346, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 11 provides the
amino acid sequences of Ceres Clone 4829 (SEQ ID NO:346), Ceres
Annot:1485102 (SEQ ID NO:350), Ceres Clone:1646533 (SEQ ID NO:351),
gi|29371519 (SEQ ID NO:352), gi|45935148 (SEQ ID NO:354), Ceres
Clone:359934 (SEQ ID NO:355), and Ceres Clone:839270 (SEQ ID
NO:357). Other homologs and/or orthologs include Ceres CLONE ID no.
24885 (SEQ ID NO:347), Ceres CLONE ID no. 27878 (SEQ ID NO:348),
Public GI no. 38347602 (SEQ ID NO:353), Ceres CLONE ID no. 294598
(SEQ ID NO:356), Ceres Clone:1836904 (SEQ ID NO:525), Ceres
Clone:1932013 (SEQ ID NO:526), and Ceres Clone:1768109 (SEQ ID
NO:527).
[0211] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:347, SEQ ID NO:348, SEQ ID
NO:350, SEQ ID NO:351, SEQ ID NO:352, SEQ ID NO:353, SEQ ID NO:354,
SEQ ID NO:355, SEQ ID NO:356, SEQ ID NO:357, SEQ ID NO:525, SEQ ID
NO:526, SEQ ID NO:527, or the consensus sequence set forth in FIG.
11.
[0212] An oil-modulating polypeptide can be a ribosomal protein
L28e. Ribosomal protein L28e forms part of the 60S ribosomal
subunit. SEQ ID NO:359 sets forth the amino acid sequence of an
Arabidopsis clone, identified herein as Ceres Clone 5426 (SEQ ID
NO:358), that is predicted to encode a ribosomal protein L28e. An
oil-modulating polypeptide can comprise the amino acid sequence set
forth in SEQ ID NO:359. Alternatively, an oil-modulating
polypeptide can be a homolog, ortholog, or variant of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:359. For example, an oil-modulating polypeptide can have an
amino acid sequence with at least 65% sequence identity, e.g., 65%,
70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity,
to the amino acid sequence set forth in SEQ ID NO:359.
[0213] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:359 are provided in FIG. 12, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:359, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 12 provides the
amino acid sequences of Ceres Clone 5426 (SEQ ID NO:359), Ceres
Clone:1123542 (SEQ ID NO:360), Ceres Annot:1499194 (SEQ ID NO:368),
and Ceres Clone:557065 (SEQ ID NO:371). Other homologs and/or
orthologs include Ceres CLONE ID no. 9083 (SEQ ID NO:361), Public
GI no. 79322493 (SEQ ID NO:362), Ceres CLONE ID no. 265408 (SEQ ID
NO:363), Ceres CLONE ID no. 32164 (SEQ ID NO:364), Ceres CLONE ID
no. 1068047 (SEQ ID NO:365), Ceres CLONE ID no. 965035 (SEQ ID
NO:366), Ceres Annot:1439584 (SEQ ID NO:370), Ceres CLONE ID no.
465060 (SEQ ID NO:372), Ceres Clone:1458107 (SEQ ID NO:512), Ceres
Clone:1932511 (SEQ ID NO:513), Ceres Clone:1850967 (SEQ ID NO:514),
Ceres Clone:1835707 (SEQ ID NO:515), Ceres Clone:1727213 (SEQ ID
NO:516), Ceres Clone:1767577 (SEQ ID NO:517), Ceres Clone:1712104
(SEQ ID NO:518), Ceres Clone:1778377 (SEQ ID NO:519), gi|76573317
(SEQ ID NO:520), Ceres Clone:1787980 (SEQ ID NO:521), gi|115465181
(SEQ ID NO:522), gi|48716267 (SEQ ID NO:523), and Ceres
Clone:575833 (SEQ ID NO:524).
[0214] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:360, SEQ ID NO:361, SEQ ID
NO:362, SEQ ID NO:363, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:366,
SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:371, SEQ ID NO:372, SEQ ID
NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516,
SEQ ID NO:517, SEQ ID NO:518, SEQ ID NO:519, SEQ ID NO:520, SEQ ID
NO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, or the
consensus sequence set forth in FIG. 12.
[0215] An oil-modulating polypeptide can be a Major Facilitator
Superfamily (MFS) transporter. MFS transporters are
single-polypeptide secondary carriers capable of transporting small
solutes in response to chemiosmotic ion gradients. SEQ ID NO:374
sets forth the amino acid sequence of an Arabidopsis clone,
identified herein as Ceres Clone 7894 (SEQ ID NO:373), that is
predicted to encode an MFS transporter polypeptide. The MFS family
of proteins is characterized by the presence of 12
membrane-spanning domains and a conserved MFS-specific motif
between membrane-spanning domains 2 and 3. Members of the MFS
protein family have been further classified into a number of
sub-families. An oil-modulating polypeptide can comprise the amino
acid sequence set forth in SEQ ID NO:374. Alternatively, an
oil-modulating polypeptide can be a homolog, ortholog, or variant
of the polypeptide having the amino acid sequence set forth in SEQ
ID NO:374. For example, an oil-modulating polypeptide can have an
amino acid sequence with at least 60% sequence identity, e.g., 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence
identity, to the amino acid sequence set forth in SEQ ID NO:374.
Such oil-modulating polypeptides may have a domain that is a
sub-family of MFS transporter polypeptides.
[0216] Amino acid sequences of homologs and/or orthologs of the
polypeptide having the amino acid sequence set forth in SEQ ID
NO:374 are provided in FIG. 13, along with a consensus sequence. A
consensus amino acid sequence for such homologs and/or orthologs
was determined by aligning amino acid sequences, e.g., amino acid
sequences related to SEQ ID NO:374, from a variety of species and
determining the most common amino acid or type of amino acid at
each position. For example, the alignment in FIG. 13 provides the
amino acid sequences of Ceres Clone 7894 (SEQ ID NO:374),
gi|18091781 (SEQ ID NO:375), gi|468562 (SEQ ID NO:376), Ceres
Annot:1479767 (SEQ ID NO:378), gi/7649151 (SEQ ID NO:379),
gi|33620334 (SEQ ID NO:382), gi|439294 (SEQ ID NO:383), gi|5230818
(SEQ ID NO:385), gi|17447420 (SEQ ID NO:386), gi|1935019 (SEQ ID
NO:387), gi|51863031 (SEQ ID NO:388), gi|575351 (SEQ ID NO:389),
gi|68161544 (SEQ ID NO:390), gi|21319 (SEQ ID NO:392), gi|5823000
(SEQ ID NO:393), gi|6120115 (SEQ ID NO:395), and gi|415988 (SEQ ID
NO:396). Other homologs and/or orthologs include Ceres
Annot:1486712 (SEQ ID NO:381), Public GI no. 77153413 (SEQ ID
NO:384), Public GI no. 6434833 (SEQ ID NO:391), Public GI no.
633172 (SEQ ID NO:394), gi|116008246 (SEQ ID NO:528), and Ceres
Clone:1925996 (SEQ ID NO:529).
[0217] In some cases, an oil-modulating polypeptide includes a
polypeptide having at least 80% sequence identity, e.g., 80%, 85%,
90%, 95%, 97%, 98%, or 99% sequence identity, to an amino acid
sequence corresponding to SEQ ID NO:375, SEQ ID NO:376, SEQ ID
NO:378, SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383,
SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID
NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392,
SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID
NO:528, SEQ ID NO:529, or the consensus sequence set forth in FIG.
13.
[0218] SEQ ID NO:398 sets forth the amino acid sequence of another
Arabidopsis clone, identified herein as Ceres Clone 8161 (SEQ ID
NO:397), that is predicted to encode a polypeptide that does not
have homology to an existing protein family based on Pfam analysis.
An oil-modulating polypeptide can comprise the amino acid sequence
set forth in SEQ ID NO:398. Alternatively, an oil-modulating
polypeptide can be a homolog having at least 40% sequence identity,
e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 98%, or 99% se quence identity, to the amino acid sequence set
forth in SEQ ID NO:398.
[0219] An oil-modulating polypeptide encoded by a recombinant
nucleic acid can be a native oil-modulating polypeptide, i.e., one
or more additional copies of the coding sequence for an
oil-modulating polypeptide that is naturally present in the cell.
Alternatively, an oil-modulating polypeptide can be heterologous to
the cell, e.g., a transgenic Lycopersicon plant can contain the
coding sequence for a transporter polypeptide from a Glycine
plant.
[0220] An oil-modulating polypeptide can include additional amino
acids that are not involved in oil modulation, and thus can be
longer than would otherwise be the case. For example, an
oil-modulating polypeptide can include an amino acid sequence that
functions as a reporter. Such an oil-modulating polypeptide can be
a fusion protein in which a green fluorescent protein (GFP)
polypeptide is fused to, e.g., SEQ ID NO:136, or in which a yellow
fluorescent protein (YFP) polypeptide is fused to, e.g., SEQ ID
NO:81. In some embodiments, an oil-modulating polypeptide includes
a purification tag, a chloroplast transit peptide, a mitochondrial
transit peptide, or a leader sequence added to the amino or carboxy
terminus.
[0221] Oil-modulating polypeptide candidates suitable for use in
the invention can be identified by analysis of nucleotide and
polypeptide sequence alignments. For example, performing a query on
a database of nucleotide or polypeptide sequences can identify
homologs and/or orthologs of oil-modulating polypeptides. Sequence
analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis
of nonredundant databases using known oil-modulating polypeptide
amino acid sequences. Those polypeptides in the database that have
greater than 40% sequence identity can be identified as candidates
for further evaluation for suitability as an oil-modulating
polypeptide. Amino acid sequence similarity allows for conservative
amino acid substitutions, such as substitution of one hydrophobic
residue for another or substitution of one polar residue for
another. If desired, manual inspection of such candidates can be
carried out in order to narrow the number of candidates to be
further evaluated. Manual inspection can be performed by selecting
those candidates that appear to have domains suspected of being
present in oil-modulating polypeptides, e.g., conserved functional
domains.
[0222] The identification of conserved regions in a template or
subject polypeptide can facilitate production of variants of wild
type oil-modulating polypeptides. Conserved regions can be
identified by locating a region within the primary amino acid
sequence of a template polypeptide that is a repeated sequence,
forms some secondary structure (e.g., helices and beta sheets),
establishes positively or negatively charged domains, or represents
a protein motif or domain. See, e.g., the Pfam web site describing
consensus sequences for a variety of protein motifs and domains on
the World Wide Web at sanger.ac.uk/Software/Pfam/ and
pfam.janelia.org/. A description of the information included at the
Pfam database is described in Sonnhammer et al., Nucl. Acids Res.,
26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997);
and Bateman et al., Nucl. Acids Res., 27:260-262 (1999).
[0223] Conserved regions also can be determined by aligning
sequences of the same or related polypeptides from closely related
species. Closely related species preferably are from the same
family. In some embodiments, alignment of sequences from two
different species is adequate. For example, sequences from
Arabidopsis and Zea mays can be used to identify one or more
conserved regions.
[0224] Typically, polypeptides that exhibit at least about 40%
amino acid sequence identity are useful to identify conserved
regions. Conserved regions of related polypeptides can exhibit at
least 45% amino acid sequence identity (e.g., at least 50%, at
least 60%, at least 70%, at least 80%, or at least 90% amino acid
sequence identity). In some embodiments, a conserved region of
target and template polypeptides exhibit at least 92%, 94%, 96%,
98%, or 99% amino acid sequence identity Amino acid sequence
identity can be deduced from amino acid or nucleotide sequences. In
certain cases, highly conserved domains have been identified within
oil-modulating polypeptides. These conserved regions can be useful
in identifying functionally similar (orthologous) oil-modulating
polypeptides.
[0225] In some instances, suitable oil-modulating polypeptides can
be synthesized on the basis of consensus functional domains and/or
conserved regions in polypeptides that are homologous
oil-modulating polypeptides. Domains are groups of substantially
contiguous amino acids in a polypeptide that can be used to
characterize protein families and/or parts of proteins. Such
domains have a "fingerprint" or "signature" that can comprise
conserved (1) primary sequence, (2) secondary structure, and/or (3)
three-dimensional conformation. Generally, domains are correlated
with specific in vitro and/or in vivo activities. A domain can have
a length of from 10 amino acids to 400 amino acids, e.g., 10 to 50
amino acids, or 25 to 100 amino acids, or 35 to 65 amino acids, or
35 to 55 amino acids, or 45 to 60 amino acids, or 200 to 300 amino
acids, or 300 to 400 amino acids.
[0226] Representative homologs and/or orthologs of oil-modulating
polypeptides are shown in FIGS. 1-13. Each Figure represents an
alignment of the amino acid sequence of an oil-modulating
polypeptide with the amino acid sequences of corresponding homologs
and/or orthologs. Amino acid sequences of oil-modulating
polypeptides and their corresponding homologs and/or orthologs have
been aligned to identify conserved amino acids and to determine
consensus sequences that contain frequently occurring amino acid
residues at particular positions in the aligned sequences, as shown
in FIGS. 1-13. A dash in an aligned sequence represents a gap,
i.e., a lack of an amino acid at that position. Identical amino
acids or conserved amino acid substitutions among aligned sequences
are identified by boxes.
[0227] Each consensus sequence is comprised of conserved regions.
Each conserved region contains a sequence of contiguous amino acid
residues. A dash in a consensus sequence indicates that the
consensus sequence either lacks an amino acid at that position or
includes an amino acid at that position. If an amino acid is
present, the residue at that position corresponds to one found in
any aligned sequence at that position.
[0228] Useful polypeptides can be constructed based on the
consensus sequence in FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG.
6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12, or FIG. 13.
Such a polypeptide includes the conserved regions in the selected
consensus sequence, arranged in the order depicted in the Figure
from amino-terminal end to carboxy-terminal end. Such a polypeptide
may also include zero, one, or more than one amino acid in
positions marked by dashes. When no amino acids are present at
positions marked by dashes, the length of such a polypeptide is the
sum of the amino acid residues in all conserved regions. When amino
acids are present at all positions marked by dashes, such a
polypeptide has a length that is the sum of the amino acid residues
in all conserved regions and all dashes.
[0229] Consensus domains and conserved regions can be identified by
homologous polypeptide sequence analysis as described above. The
suitability of polypeptides for use as oil-modulating polypeptides
can be evaluated by functional complementation studies.
Nucleic Acids
[0230] Isolated nucleic acids are provided herein. The terms
"nucleic acid" and "polynucleotide" are used interchangeably
herein, and refer to both RNA and DNA, including cDNA, genomic DNA,
synthetic DNA, and DNA (or RNA) containing nucleic acid analogs.
Polynucleotides can have any three-dimensional structure. A nucleic
acid can be double-stranded or single-stranded (i.e., a sense
strand or an antisense strand). Non-limiting examples of
polynucleotides include genes, gene fragments, exons, introns,
messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA,
micro-RNA, ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, nucleic acid probes, and primers, as
well as nucleic acid analogs.
[0231] Nucleic acids described herein include oil-modulating
nucleic acids. Oil-modulating nucleic acids can be effective to
modulate protein levels when transcribed in a plant or plant cell.
A oil-modulating nucleic acid can comprise the nucleotide sequence
set forth in SEQ ID NO:80, SEQ ID NO:93, SEQ ID NO:95, SEQ ID
NO:108, SEQ ID NO:110, SEQ ID NO:117, SEQ ID NO:135, SEQ ID NO:141,
SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:151, SEQ ID
NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:170, SEQ ID NO:173,
SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:180, SEQ ID NO:183, SEQ ID
NO:192, SEQ ID NO:197, SEQ ID NO:218, SEQ ID NO:221, SEQ ID NO:230,
SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:251, SEQ ID NO:331, SEQ ID
NO:334, SEQ ID NO:336, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345,
SEQ ID NO:349, SEQ ID NO:358, SEQ ID NO:367, SEQ ID NO:369, SEQ ID
NO:373, SEQ ID NO:377, SEQ ID NO:380, SEQ ID NO:397, SEQ ID NO:399,
SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:403, SEQ ID
NO:404, SEQ ID NO:405, SEQ ID NO:406, SEQ ID NO:407, SEQ ID NO:408,
SEQ ID NO:409, SEQ ID NO:410, SEQ ID NO:411, SEQ ID NO:412, SEQ ID
NO:413, SEQ ID NO:414, SEQ ID NO:415, SEQ ID NO:416, SEQ ID NO:417,
SEQ ID NO:418, SEQ ID NO:419, SEQ ID NO:420, SEQ ID NO:421, SEQ ID
NO:422, SEQ ID NO:423, SEQ ID NO:424, SEQ ID NO:425, SEQ ID NO:426,
SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID
NO:431, SEQ ID NO:432, SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435,
SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID
NO:440, SEQ ID NO:441, SEQ ID NO:442, SEQ ID NO:443, SEQ ID NO:444,
SEQ ID NO:445, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ ID
NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, SEQ ID NO:453,
SEQ ID NO:454, SEQ ID NO:455, SEQ ID NO:456, SEQ ID NO:457, SEQ ID
NO:458, SEQ ID NO:459, SEQ ID NO:460, SEQ ID NO:461, SEQ ID NO:462,
SEQ ID NO:463, SEQ ID NO:464, SEQ ID NO:465, SEQ ID NO:466, SEQ ID
NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471,
SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:474, SEQ ID NO:475, SEQ ID
NO:476, SEQ ID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480,
SEQ ID NO:481, SEQ ID NO:482, SEQ ID NO:483, SEQ ID NO:484, SEQ ID
NO:485, SEQ ID NO:486, SEQ ID NO:487, SEQ ID NO:488, SEQ ID NO:489,
SEQ ID NO:490, SEQ ID NO:491, SEQ ID NO:492, SEQ ID NO:493, SEQ ID
NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:498,
SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:546, SEQ ID
NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551,
SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID
NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560,
SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID
NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569,
SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID
NO:574, or SEQ ID NO:575. Alternatively, a oil-modulating nucleic
acid can be a variant of the nucleic acid having the nucleotide
sequence set forth in SEQ ID NO:80, SEQ ID NO:93, SEQ ID NO:95, SEQ
ID NO:108, SEQ ID NO:110, SEQ ID NO:117, SEQ ID NO:135, SEQ ID
NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:151,
SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:170, SEQ ID
NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:180, SEQ ID NO:183,
SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:218, SEQ ID NO:221, SEQ ID
NO:230, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:251, SEQ ID NO:331,
SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:341, SEQ ID NO:343, SEQ ID
NO:345, SEQ ID NO:349, SEQ ID NO:358, SEQ ID NO:367, SEQ ID NO:369,
SEQ ID NO:373, SEQ ID NO:377, SEQ ID NO:380, SEQ ID NO:397, SEQ ID
NO:399, SEQ ID NO:400, SEQ ID NO:401, SEQ ID NO:402, SEQ ID NO:403,
SEQ ID NO:404, SEQ ID NO:405, SEQ ID NO:406, SEQ ID NO:407, SEQ ID
NO:408, SEQ ID NO:409, SEQ ID NO:410, SEQ ID NO:411, SEQ ID NO:412,
SEQ ID NO:413, SEQ ID NO:414, SEQ ID NO:415, SEQ ID NO:416, SEQ ID
NO:417, SEQ ID NO:418, SEQ ID NO:419, SEQ ID NO:420, SEQ ID NO:421,
SEQ ID NO:422, SEQ ID NO:423, SEQ ID NO:424, SEQ ID NO:425, SEQ ID
NO:426, SEQ ID NO:427, SEQ ID NO:428, SEQ ID NO:429, SEQ ID NO:430,
SEQ ID NO:431, SEQ ID NO:432, SEQ ID NO:433, SEQ ID NO:434, SEQ ID
NO:435, SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439,
SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:442, SEQ ID NO:443, SEQ ID
NO:444, SEQ ID NO:445, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448,
SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, SEQ ID
NO:453, SEQ ID NO:454, SEQ ID NO:455, SEQ ID NO:456, SEQ ID NO:457,
SEQ ID NO:458, SEQ ID NO:459, SEQ ID NO:460, SEQ ID NO:461, SEQ ID
NO:462, SEQ ID NO:463, SEQ ID NO:464, SEQ ID NO:465, SEQ ID NO:466,
SEQ ID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:470, SEQ ID
NO:471, SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:474, SEQ ID NO:475,
SEQ ID NO:476, SEQ ID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID
NO:480, SEQ ID NO:481, SEQ ID NO:482, SEQ ID NO:483, SEQ ID NO:484,
SEQ ID NO:485, SEQ ID NO:486, SEQ ID NO:487, SEQ ID NO:488, SEQ ID
NO:489, SEQ ID NO:490, SEQ ID NO:491, SEQ ID NO:492, SEQ ID NO:493,
SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID
NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:546,
SEQ ID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID
NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555,
SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID
NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564,
SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID
NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573,
SEQ ID NO:574, or SEQ ID NO:575. For example, a oil-modulating
nucleic acid can have a nucleotide sequence with at least 80%
sequence identity, e.g., 81%, 85%, 90%, 95%, 97%, 98%, or 99%
sequence identity, to the nucleotide sequence set forth in SEQ ID
NO:80, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:108, SEQ ID NO:110,
SEQ ID NO:117, SEQ ID NO:135, SEQ ID NO:141, SEQ ID NO:143, SEQ ID
NO:145, SEQ ID NO:148, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,
SEQ ID NO:158, SEQ ID NO:170, SEQ ID NO:173, SEQ ID NO:175, SEQ ID
NO:177, SEQ ID NO:180, SEQ ID NO:183, SEQ ID NO:192, SEQ ID NO:197,
SEQ ID NO:218, SEQ ID NO:221, SEQ ID NO:230, SEQ ID NO:239, SEQ ID
NO:241, SEQ ID NO:251, SEQ ID NO:331, SEQ ID NO:334, SEQ ID NO:336,
SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:349, SEQ ID
NO:358, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:373, SEQ ID NO:377,
SEQ ID NO:380, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:400, SEQ ID
NO:401, SEQ ID NO:402, SEQ ID NO:403, SEQ ID NO:404, SEQ ID NO:405,
SEQ ID NO:406, SEQ ID NO:407, SEQ ID NO:408, SEQ ID NO:409, SEQ ID
NO:410, SEQ ID NO:411, SEQ ID NO:412, SEQ ID NO:413, SEQ ID NO:414,
SEQ ID NO:415, SEQ ID NO:416, SEQ ID NO:417, SEQ ID NO:418, SEQ ID
NO:419, SEQ ID NO:420, SEQ ID NO:421, SEQ ID NO:422, SEQ ID NO:423,
SEQ ID NO:424, SEQ ID NO:425, SEQ ID NO:426, SEQ ID NO:427, SEQ ID
NO:428, SEQ ID NO:429, SEQ ID NO:430, SEQ ID NO:431, SEQ ID NO:432,
SEQ ID NO:433, SEQ ID NO:434, SEQ ID NO:435, SEQ ID NO:436, SEQ ID
NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:441,
SEQ ID NO:442, SEQ ID NO:443, SEQ ID NO:444, SEQ ID NO:445, SEQ ID
NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:449, SEQ ID NO:450,
SEQ ID NO:451, SEQ ID NO:452, SEQ ID NO:453, SEQ ID NO:454, SEQ ID
NO:455, SEQ ID NO:456, SEQ ID NO:457, SEQ ID NO:458, SEQ ID NO:459,
SEQ ID NO:460, SEQ ID NO:461, SEQ ID NO:462, SEQ ID NO:463, SEQ ID
NO:464, SEQ ID NO:465, SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468,
SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472, SEQ ID
NO:473, SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476, SEQ ID NO:477,
SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480, SEQ ID NO:481, SEQ ID
NO:482, SEQ ID NO:483, SEQ ID NO:484, SEQ ID NO:485, SEQ ID NO:486,
SEQ ID NO:487, SEQ ID NO:488, SEQ ID NO:489, SEQ ID NO:490, SEQ ID
NO:491, SEQ ID NO:492, SEQ ID NO:493, SEQ ID NO:494, SEQ ID NO:495,
SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID
NO:500, SEQ ID NO:501, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548,
SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID
NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557,
SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID
NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566,
SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID
NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, or SEQ ID
NO:575.
[0232] An "isolated nucleic acid" can be, for example, a
naturally-occurring DNA molecule, provided one of the nucleic acid
sequences normally found immediately flanking that DNA molecule in
a naturally-occurring genome is removed or absent. Thus, an
isolated nucleic acid includes, without limitation, a DNA molecule
that exists as a separate molecule, independent of other sequences
(e.g., a chemically synthesized nucleic acid, or a cDNA or genomic
DNA fragment produced by the polymerase chain reaction (PCR) or
restriction endonuclease treatment). An isolated nucleic acid also
refers to a DNA molecule that is incorporated into a vector, an
autonomously replicating plasmid, a virus, or into the genomic DNA
of a prokaryote or eukaryote. In addition, an isolated nucleic acid
can include an engineered nucleic acid such as a DNA molecule that
is part of a hybrid or fusion nucleic acid. A nucleic acid existing
among hundreds to millions of other nucleic acids within, for
example, cDNA libraries or genomic libraries, or gel slices
containing a genomic DNA restriction digest, is not to be
considered an isolated nucleic acid.
[0233] Isolated nucleic acid molecules can be produced by standard
techniques. For example, polymerase chain reaction (PCR) techniques
can be used to obtain an isolated nucleic acid containing a
nucleotide sequence described herein. PCR can be used to amplify
specific sequences from DNA as well as RNA, including sequences
from total genomic DNA or total cellular RNA. Various PCR methods
are described, for example, in PCR Primer: A Laboratory Manual,
Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory
Press, 1995. Generally, sequence information from the ends of the
region of interest or beyond is employed to design oligonucleotide
primers that are identical or similar in sequence to opposite
strands of the template to be amplified. Various PCR strategies
also are available by which site-specific nucleotide sequence
modifications can be introduced into a template nucleic acid.
Isolated nucleic acids also can be chemically synthesized, either
as a single nucleic acid molecule (e.g., using automated DNA
synthesis in the 3' to 5' direction using phosphoramidite
technology) or as a series of oligonucleotides. For example, one or
more pairs of long oligonucleotides (e.g., >100 nucleotides) can
be synthesized that contain the desired sequence, with each pair
containing a short segment of complementarity (e.g., about 15
nucleotides) such that a duplex is formed when the oligonucleotide
pair is annealed. DNA polymerase is used to extend the
oligonucleotides, resulting in a single, double-stranded nucleic
acid molecule per oligonucleotide pair, which then can be ligated
into a vector. Isolated nucleic acids of the invention also can be
obtained by mutagenesis of, e.g., a naturally occurring DNA.
[0234] As used herein, the term "percent sequence identity" refers
to the degree of identity between any given query sequence, e.g.,
SEQ ID NO:81, and a subject sequence. A subject sequence typically
has a length that is from 80 percent to 200 percent of the length
of the query sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99,
100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200
percent of the length of the query sequence. A percent identity for
any subject nucleic acid or polypeptide relative to a query nucleic
acid or polypeptide can be determined as follows. A query sequence
(e.g., a nucleic acid sequence or an amino acid sequence) is
aligned to one or more subject sequences using the computer program
ClustalW (version 1.83, default parameters), which allows
alignments of nucleic acid or polypeptide sequences to be carried
out across their entire length (global alignment). Chema et al.,
Nucleic Acids Res., 31(13):3497-500 (2003).
[0235] ClustalW calculates the best match between a query and one
or more subject sequences, and aligns them so that identities,
similarities and differences can be determined Gaps of one or more
residues can be inserted into a query sequence, a subject sequence,
or both, to maximize sequence alignments. For fast pairwise
alignment of nucleic acid sequences, the following default
parameters are used: word size: 2; window size: 4; scoring method:
percentage; number of top diagonals: 4; and gap penalty: 5. For
multiple alignment of nucleic acid sequences, the following
parameters are used: gap opening penalty: 10.0; gap extension
penalty: 5.0; and weight transitions: yes. For fast pairwise
alignment of protein sequences, the following parameters are used:
word size: 1; window size: 5; scoring method: percentage; number of
top diagonals: 5; gap penalty: 3. For multiple alignment of protein
sequences, the following parameters are used: weight matrix:
blosum; gap opening penalty: 10.0; gap extension penalty: 0.05;
hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn,
Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on.
The ClustalW output is a sequence alignment that reflects the
relationship between sequences. ClustalW can be run, for example,
at the Baylor College of Medicine Search Launcher site
(searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at
the European Bioinformatics Institute site on the World Wide Web
(ebi.ac.uk/clustalw).
[0236] To determine percent identity of a subject nucleic acid or
amino acid sequence to a query sequence, the sequences are aligned
using ClustalW, the number of identical matches in the alignment is
divided by the length of the query sequence, and the result is
multiplied by 100. It is noted that the percent identity value can
be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13,
and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17,
78.18, and 78.19 are rounded up to 78.2.
[0237] The term "exogenous" with respect to a nucleic acid
indicates that the nucleic acid is part of a recombinant nucleic
acid construct, or is not in its natural environment. For example,
an exogenous nucleic acid can be a sequence from one species
introduced into another species, i.e., a heterologous nucleic acid.
Typically, such an exogenous nucleic acid is introduced into the
other species via a recombinant nucleic acid construct. An
exogenous nucleic acid can also be a sequence that is native to an
organism and that has been reintroduced into cells of that
organism. An exogenous nucleic acid that includes a native sequence
can often be distinguished from the naturally occurring sequence by
the presence of non-natural sequences linked to the exogenous
nucleic acid, e.g., non-native regulatory sequences flanking a
native sequence in a recombinant nucleic acid construct. In
addition, stably transformed exogenous nucleic acids typically are
integrated at positions other than the position where the native
sequence is found. It will be appreciated that an exogenous nucleic
acid may have been introduced into a progenitor and not into the
cell under consideration. For example, a transgenic plant
containing an exogenous nucleic acid can be the progeny of a cross
between a stably transformed plant and a non-transgenic plant. Such
progeny are considered to contain the exogenous nucleic acid.
[0238] Recombinant constructs are also provided herein and can be
used to transform plants or plant cells in order to modulate oil
levels. A recombinant nucleic acid construct comprises a nucleic
acid encoding an oil-modulating polypeptide as described herein,
operably linked to a regulatory region suitable for expressing the
oil-modulating polypeptide in the plant or cell. Thus, a nucleic
acid can comprise a coding sequence that encodes any of the
oil-modulating polypeptides as set forth in SEQ ID NOs:81-92, SEQ
ID NO:94, SEQ ID NOs:96-107, SEQ ID NO:109, SEQ ID NOs:111-116, SEQ
ID NOs:118-134, SEQ ID NOs:136-140, SEQ ID NO:142, SEQ ID NO:144,
SEQ ID NOs:146-147, SEQ ID NOs:149-150, SEQ ID NO:152, SEQ ID
NO:154, SEQ ID NOs:156-157, SEQ ID NOs:159-169, SEQ ID NOs:171-172,
SEQ ID NO:174, SEQ ID NO:176, SEQ ID NOs:178-179, SEQ ID
NOs:181-182, SEQ ID NOs:184-191, SEQ ID NOs:193-196, SEQ ID
NOs:198-217, SEQ ID NOs:219-220, SEQ ID NOs:222-229, SEQ ID
NOs:231-238, SEQ ID NO:240, SEQ ID NOs:242-250, SEQ ID NOs:252-330,
SEQ ID NOs:332-333, SEQ ID NO:335, SEQ ID NOs:337-340, SEQ ID
NO:342, SEQ ID NO:344, SEQ ID NO:346-348, SEQ ID NOs:350-357, SEQ
ID NOs:359-366, SEQ ID NO:368, SEQ ID NOs:370-372, SEQ ID
NOs:374-376, SEQ ID NOs:378-379, SEQ ID NOs:381-396, SEQ ID NO:398,
SEQ ID NOs:502-545, and the consensus sequences set forth in FIGS.
1-13. Examples of nucleic acids encoding oil-modulating
polypeptides are set forth in SEQ ID NO:80, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:117, SEQ ID NO:135,
SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:148, SEQ ID
NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:170,
SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:180, SEQ ID
NO:183, SEQ ID NO:192, SEQ ID NO:197, SEQ ID NO:218, SEQ ID NO:221,
SEQ ID NO:230, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:251, SEQ ID
NO:331, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:341, SEQ ID NO:343,
SEQ ID NO:345, SEQ ID NO:349, SEQ ID NO:358, SEQ ID NO:367, SEQ ID
NO:369, SEQ ID NO:373, SEQ ID NO:377, SEQ ID NO:380, SEQ ID NO:397,
SEQ ID NOs:399-501, and SEQ ID NOs:546-575.
[0239] In some cases, a recombinant nucleic acid construct can
include a nucleic acid comprising less than the full-length of a
coding sequence. Typically, such a construct also includes a
regulatory region operably linked to the oil-modulating nucleic
acid. In some cases, a recombinant nucleic acid construct can
include a nucleic acid comprising a coding sequence, a gene, or a
fragment of a coding sequence or gene in an antisense orientation
so that the antisense strand of RNA is transcribed.
[0240] It will be appreciated that a number of nucleic acids can
encode a polypeptide having a particular amino acid sequence. The
degeneracy of the genetic code is well known to the art; i.e., for
many amino acids, there is more than one nucleotide triplet that
serves as the codon for the amino acid. For example, codons in the
coding sequence for a given oil-modulating polypeptide can be
modified such that optimal expression in a particular plant species
is obtained, using appropriate codon bias tables for that
species.
[0241] Vectors containing nucleic acids such as those described
herein also are provided. A "vector" is a replicon, such as a
plasmid, phage, or cosmid, into which another DNA segment may be
inserted so as to bring about the replication of the inserted
segment. Generally, a vector is capable of replication when
associated with the proper control elements. Suitable vector
backbones include, for example, those routinely used in the art
such as plasmids, viruses, artificial chromosomes, BACs, YACs, or
PACs. The term "vector" includes cloning and expression vectors, as
well as viral vectors and integrating vectors. An "expression
vector" is a vector that includes a regulatory region. Suitable
expression vectors include, without limitation, plasmids and viral
vectors derived from, for example, bacteriophage, baculoviruses,
and retroviruses. Numerous vectors and expression systems are
commercially available from such corporations as Novagen (Madison,
Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.),
and Invitrogen/Life Technologies (Carlsbad, Calif.).
[0242] The vectors provided herein also can include, for example,
origins of replication, scaffold attachment regions (SARs), and/or
markers. A marker gene can confer a selectable phenotype on a plant
cell. For example, a marker can confer biocide resistance, such as
resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or
hygromycin), or an herbicide (e.g., chlorosulfuron or
phosphinothricin). In addition, an expression vector can include a
tag sequence designed to facilitate manipulation or detection
(e.g., purification or localization) of the expressed polypeptide.
Tag sequences, such as green fluorescent protein (GFP), glutathione
S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or
Flag.TM. tag (Kodak, New Haven, Conn.) sequences typically are
expressed as a fusion with the encoded polypeptide. Such tags can
be inserted anywhere within the polypeptide, including at either
the carboxyl or amino terminus
Regulatory Regions
[0243] The term "regulatory region" refers to nucleotide sequences
that influence transcription or translation initiation and rate,
and stability and/or mobility of a transcription or translation
product. Regulatory regions include, without limitation, promoter
sequences, enhancer sequences, response elements, protein
recognition sites, inducible elements, protein binding sequences,
5' and 3' untranslated regions (UTRs), transcriptional start sites,
termination sequences, polyadenylation sequences, introns, and
combinations thereof.
[0244] As used herein, the term "operably linked" refers to
positioning of a regulatory region and a sequence to be transcribed
in a nucleic acid so as to influence transcription or translation
of such a sequence. For example, to bring a coding sequence under
the control of a promoter, the translation initiation site of the
translational reading frame of the polypeptide is typically
positioned between one and about fifty nucleotides downstream of
the promoter. A promoter can, however, be positioned as much as
about 5,000 nucleotides upstream of the translation initiation
site, or about 2,000 nucleotides upstream of the transcription
start site. A promoter typically comprises at least a core (basal)
promoter. A promoter also may include at least one control element,
such as an enhancer sequence, an upstream element or an upstream
activation region (UAR). For example, a suitable enhancer is a
cis-regulatory element (-212 to -154) from the upstream region of
the octopine synthase (ocs) gene. Fromm et al., The Plant Cell,
1:977-984 (1989). The choice of promoters to be included depends
upon several factors, including, but not limited to, efficiency,
selectability, inducibility, desired expression level, and cell- or
tissue-preferential expression. It is a routine matter for one of
skill in the art to modulate the expression of a coding sequence by
appropriately selecting and positioning regulatory regions relative
to the coding sequence.
[0245] Some suitable regulatory regions initiate transcription
only, or predominantly, in certain cell types, for example, a
promoter that is active predominantly in a reproductive tissue such
as fruit, ovule, pollen, pistils, female gametophyte, egg cell,
central cell, nucellus, suspensor, synergid cell, flowers,
embryonic tissue, embryo sac, embryo, zygote, endosperm,
integument, or seed coat. Thus, as used herein a cell type- or
tissue-preferential promoter is one that drives expression
preferentially in the target tissue, but may also lead to some
expression in other cell types or tissues as well. Methods for
identifying and characterizing promoter regions in plant genomic
DNA include, for example, those described in the following
references: Jordano et al., Plant Cell, 1: 855-866 (1989); Bustos
et al., Plant Cell, 1:839-854 (1989); Green et al., EMBO J.,
7:4035-4044 (1988); Meier et al., Plant Cell, 3:309-316 (1991); and
Zhang et al., Plant Physiology, 110:1069-1079 (1996).
[0246] Examples of various classes of regulatory regions are
described below. Some of the regulatory regions indicated below as
well as additional regulatory regions are described in more detail
in U.S. Pat. Application Ser. Nos. 60/505,689; 60/518,075;
60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140;
60/757,544; 60/776,307; 10/957,569; 11/058,689; 11/172,703;
11/208,308; 11/274,890; 60/583,609; 60/612,891; 11/097,589;
11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017;
PCT/US05/011105; PCT/US05/034308; and PCT/US05/23639. Nucleotide
sequences of promoters are set forth in SEQ ID NOs:1-79 and
259-274. It will be appreciated that a regulatory region may meet
criteria for one classification based on its activity in one plant
species, and yet meet criteria for a different classification based
on its activity in another plant species.
[0247] Broadly Expressing Promoters
[0248] A promoter can be said to be "broadly expressing" when it
promotes transcription in many, but not necessarily all, plant
tissues. For example, a broadly expressing promoter can promote
transcription of an operably linked sequence in one or more of the
shoot, shoot tip (apex), and leaves, but weakly or not at all in
tissues such as roots or stems. As another example, a broadly
expressing promoter can promote transcription of an operably linked
sequence in one or more of the stem, shoot, shoot tip (apex), and
leaves, but can promote transcription weakly or not at all in
tissues such as reproductive tissues of flowers and developing
seeds. Non-limiting examples of broadly expressing promoters that
can be included in the nucleic acid constructs provided herein
include the p326 (SEQ ID NO:76), YP0144 (SEQ ID NO:55), YP0190 (SEQ
ID NO:59), p13879 (SEQ ID NO:75), YP0050 (SEQ ID NO:35), p32449
(SEQ ID NO:77), 21876 (SEQ ID NO:1), YP0158 (SEQ ID NO:57), YP0214
(SEQ ID NO:61), YP0380 (SEQ ID NO:70), PT0848 (SEQ ID NO:26), and
PT0633 (SEQ ID NO:7) promoters. Additional examples include the
cauliflower mosaic virus (CaMV) 35S promoter, the mannopine
synthase (MAS) promoter, the 1' or 2' promoters derived from T-DNA
of Agrobacterium tumefaciens, the figwort mosaic virus 34S
promoter, actin promoters such as the rice actin promoter, and
ubiquitin promoters such as the maize ubiquitin-1 promoter. In some
cases, the CaMV 35S promoter is excluded from the category of
broadly expressing promoters.
[0249] Root Promoters
[0250] Root-active promoters confer transcription in root tissue,
e.g., root endodermis, root epidermis, or root vascular tissues. In
some embodiments, root-active promoters are root-preferential
promoters, i.e., confer transcription only or predominantly in root
tissue. Root-preferential promoters include the YP0128 (SEQ ID
NO:52), YP0275 (SEQ ID NO:63), PT0625 (SEQ ID NO:6), PT0660 (SEQ ID
NO:9), PT0683 (SEQ ID NO:14), and PT0758 (SEQ ID NO:22) promoters.
Other root-preferential promoters include the PT0613 (SEQ ID NO:5),
PT0672 (SEQ ID NO:11), PT0688 (SEQ ID NO:15), and PT0837 (SEQ ID
NO:24) promoters, which drive transcription primarily in root
tissue and to a lesser extent in ovules and/or seeds. Other
examples of root-preferential promoters include the root-specific
subdomains of the CaMV 35S promoter (Lam et al., Proc. Natl. Acad.
Sci. USA, 86:7890-7894 (1989)), root cell specific promoters
reported by Conkling et al., Plant Physiol., 93:1203-1211 (1990),
and the tobacco RD2 promoter.
[0251] Maturing Endosperm Promoters
[0252] In some embodiments, promoters that drive transcription in
maturing endosperm can be useful. Transcription from a maturing
endosperm promoter typically begins after fertilization and occurs
primarily in endosperm tissue during seed development and is
typically highest during the cellularization phase. Most suitable
are promoters that are active predominantly in maturing endosperm,
although promoters that are also active in other tissues can
sometimes be used. Non-limiting examples of maturing endosperm
promoters that can be included in the nucleic acid constructs
provided herein include the napin promoter, the Arcelin-5 promoter,
the phaseolin promoter (Bustos et al., Plant Cell, 1(9):839-853
(1989)), the soybean trypsin inhibitor promoter (Riggs et al.,
Plant Cell, 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,
Plant Mol. Biol., 22(2):255-267 (1993)), the stearoyl-ACP
desaturase promoter (Slocombe et al., Plant Physiol.,
104(4):167-176 (1994)), the soybean .alpha.' subunit of
.beta.-conglycinin promoter (Chen et al., Proc. Natl. Acad. Sci.
USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al., Plant
Mol. Biol., 34(3):549-555 (1997)), and zein promoters, such as the
15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter,
22 kD zein promoter and 27 kD zein promoter. Also suitable are the
Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al., Mol.
Cell. Biol., 13:5829-5842 (1993)), the beta-amylase promoter, and
the barley hordein promoter. Other maturing endosperm promoters
include the YP0092 (SEQ ID NO:38), PT0676 (SEQ ID NO:12), and
PT0708 (SEQ ID NO:17) promoters.
[0253] Ovary Tissue Promoters
[0254] Promoters that are active in ovary tissues such as the ovule
wall and mesocarp can also be useful, e.g., a polygalacturonidase
promoter, the banana TRX promoter, the melon actin promoter, YP0396
(SEQ ID NO:74), and PT0623 (SEQ ID NO:273). Examples of promoters
that are active primarily in ovules include YP0007 (SEQ ID NO:30),
YP0111 (SEQ ID NO:46), YP0092 (SEQ ID NO:38), YP0103 (SEQ ID
NO:43), YP0028 (SEQ ID NO:33), YP0121 (SEQ ID NO:51), YP0008 (SEQ
ID NO:31), YP0039 (SEQ ID NO:34), YP0115 (SEQ ID NO:47), YP0119
(SEQ ID NO:49), YP0120 (SEQ ID NO:50), and YP0374 (SEQ ID
NO:68).
[0255] Embryo Sac/Early Endosperm Promoters
[0256] To achieve expression in embryo sac/early endosperm,
regulatory regions can be used that are active in polar nuclei
and/or the central cell, or in precursors to polar nuclei, but not
in egg cells or precursors to egg cells. Most suitable are
promoters that drive expression only or predominantly in polar
nuclei or precursors thereto and/or the central cell. A pattern of
transcription that extends from polar nuclei into early endosperm
development can also be found with embryo sac/early
endosperm-preferential promoters, although transcription typically
decreases significantly in later endosperm development during and
after the cellularization phase. Expression in the zygote or
developing embryo typically is not present with embryo sac/early
endosperm promoters.
[0257] Promoters that may be suitable include those derived from
the following genes: Arabidopsis viviparous-1 (see, GenBank No.
U93215); Arabidopsis atmycl (see, Urao (1996) Plant Mol. Biol.,
32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE
(GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank
No. AF096096); and FIE 1.1 (U.S. Pat. No. 6,906,244). Other
promoters that may be suitable include those derived from the
following genes: maize MAC1 (see, Sheridan (1996) Genetics,
142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993)
Plant Mol. Biol., 22:10131-1038). Other promoters include the
following Arabidopsis promoters: YP0039 (SEQ ID NO:34), YP0101 (SEQ
ID NO:41), YP0102 (SEQ ID NO:42), YP0110 (SEQ ID NO:45), YP0117
(SEQ ID NO:48), YP0119 (SEQ ID NO:49), YP0137 (SEQ ID NO:53), DME,
YP0285 (SEQ ID NO:64), and YP0212 (SEQ ID NO:60). Other promoters
that may be useful include the following rice promoters: p530c10
(SEQ ID NO:576), pOsFIE2-2 (SEQ ID NO:577), pOsMEA (SEQ ID NO:578),
pOsYp102 (SEQ ID NO:579), and pOsYp285 (SEQ ID NO:580).
[0258] Embryo Promoters
[0259] Regulatory regions that preferentially drive transcription
in zygotic cells following fertilization can provide
embryo-preferential expression. Most suitable are promoters that
preferentially drive transcription in early stage embryos prior to
the heart stage, but expression in late stage and maturing embryos
is also suitable. Embryo-preferential promoters include the barley
lipid transfer protein (Ltp1) promoter (Plant Cell Rep (2001)
20:647-654), YP0097 (SEQ ID NO:40), YP0107 (SEQ ID NO:44), YP0088
(SEQ ID NO:37), YP0143 (SEQ ID NO:54), YP0156 (SEQ ID NO:56),
PT0650 (SEQ ID NO:8), PT0695 (SEQ ID NO:16), PT0723 (SEQ ID NO:19),
PT0838 (SEQ ID NO:25), PT0879 (SEQ ID NO:28), and PT0740 (SEQ ID
NO:20).
[0260] Photosynthetic Tissue Promoters
[0261] Promoters active in photosynthetic tissue confer
transcription in green tissues such as leaves and stems. Most
suitable are promoters that drive expression only or predominantly
in such tissues. Examples of such promoters include the
ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the
RbcS promoter from eastern larch (Larix laricina), the pine cab6
promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778 (1994)),
the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol.,
15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et
al., Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from
rice (Luan et al., Plant Cell, 4:971-981 (1992)), the pyruvate
orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al.,
Proc. Natl. Acad. Sci. USA, 90:9586-9590 (1993)), the tobacco
Lhcb1*2 promoter (Cerdan et al., Plant Mol. Biol., 33:245-255
(1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporter
promoter (Truernit et al., Planta, 196:564-570 (1995)), and
thylakoid membrane protein promoters from spinach (psaD, psaF,
psaE, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissue
promoters include PT0535 (SEQ ID NO:3), PT0668 (SEQ ID NO:2),
PT0886 (SEQ ID NO:29), PRO924 (SEQ ID NO:78), YP0144 (SEQ ID
NO:55), YP0380 (SEQ ID NO:70), and PT0585 (SEQ ID NO:4).
[0262] Vascular Tissue Promoters
[0263] Examples of promoters that have high or preferential
activity in vascular bundles include YP0087 (SEQ ID NO:583), YP0093
(SEQ ID NO:584), YP0108 (SEQ ID NO:585), YP0022 (SEQ ID NO:586),
and YP0080 (SEQ ID NO:587). Other vascular tissue-preferential
promoters include the glycine-rich cell wall protein GRP 1.8
promoter (Keller and Baumgartner, Plant Cell, 3(10):1051-1061
(1991)), the Commelina yellow mottle virus (CoYMV) promoter
(Medberry et al., Plant Cell, 4(2):185-192 (1992)), and the rice
tungro bacilliform virus (RTBV) promoter (Dai et al., Proc. Natl.
Acad. Sci. USA, 101(2):687-692 (2004)).
[0264] Inducible Promoters
[0265] Inducible promoters confer transcription in response to
external stimuli such as chemical agents or environmental stimuli.
For example, inducible promoters can confer transcription in
response to hormones such as giberellic acid or ethylene, or in
response to light or drought. Examples of drought-inducible
promoters include YP0380 (SEQ ID NO:70), PT0848 (SEQ ID NO:26),
YP0381 (SEQ ID NO:71), YP0337 (SEQ ID NO:66), PT0633 (SEQ ID NO:7),
YP0374 (SEQ ID NO:68), PT0710 (SEQ ID NO:18), YP0356 (SEQ ID
NO:67), YP0385 (SEQ ID NO:73), YP0396 (SEQ ID NO:74), YP0388 (SEQ
ID NO:588), YP0384 (SEQ ID NO:72), PT0688 (SEQ ID NO:15), YP0286
(SEQ ID NO:65), YP0377 (SEQ ID NO:69), PD1367 (SEQ ID NO:79), and
PD0901 (SEQ ID NO:589. Nitrogen-inducible promoters include PT0863
(SEQ ID NO:27), PT0829 (SEQ ID NO:23), PT0665 (SEQ ID NO:10), and
PT0886 (SEQ ID NO:29). Examples of a shade-inducible promoters are
PRO924 (SEQ ID NO:78) and PT0678 (SEQ ID NO:13).
[0266] Basal Promoters
[0267] A basal promoter is the minimal sequence necessary for
assembly of a transcription complex required for transcription
initiation. Basal promoters frequently include a "TATA box" element
that may be located between about 15 and about 35 nucleotides
upstream from the site of transcription initiation. Basal promoters
also may include a "CCAAT box" element (typically the sequence
CCAAT) and/or a GGGCG sequence, which can be located between about
40 and about 200 nucleotides, typically about 60 to about 120
nucleotides, upstream from the transcription start site.
[0268] Other Promoters
[0269] Other classes of promoters include, but are not limited to,
leaf-preferential, stem/shoot-preferential, callus-preferential,
guard cell-preferential, such as PT0678 (SEQ ID NO:13),
tuber-preferential, parenchyma cell-preferential, and
senescence-preferential promoters. Promoters designated YP0086 (SEQ
ID NO:36), YP0188 (SEQ ID NO:58), YP0263 (SEQ ID NO:62), PT0758
(SEQ ID NO:22), PT0743 (SEQ ID NO:21), PT0829 (SEQ ID NO:23),
YP0119 (SEQ ID NO:49), and YP0096 (SEQ ID NO:39), as described in
the above-referenced patent applications, may also be useful.
[0270] Other Regulatory Regions
[0271] A 5' untranslated region (UTR) can be included in nucleic
acid constructs described herein. A 5' UTR is transcribed, but is
not translated, and lies between the start site of the transcript
and the translation initiation codon and may include the +1
nucleotide. A 3' UTR can be positioned between the translation
termination codon and the end of the transcript. UTRs can have
particular functions such as increasing mRNA stability or
attenuating translation. Examples of 3' UTRs include, but are not
limited to, polyadenylation signals and transcription termination
sequences, e.g., a nopaline synthase termination sequence.
[0272] It will be understood that more than one regulatory region
may be present in a recombinant polynucleotide, e.g., introns,
enhancers, upstream activation regions, transcription terminators,
and inducible elements. Thus, for example, more than one regulatory
region can be operably linked to the sequence of a polynucleotide
encoding an oil-modulating polypeptide.
[0273] Regulatory regions, such as promoters for endogenous genes,
can be obtained by chemical synthesis or by subcloning from a
genomic DNA that includes such a regulatory region. A nucleic acid
comprising such a regulatory region can also include flanking
sequences that contain restriction enzyme sites that facilitate
subsequent manipulation.
Transgenic Plants and Plant Cells
[0274] The invention also features transgenic plant cells and
plants comprising at least one recombinant nucleic acid construct
described herein. A plant or plant cell can be transformed by
having a construct integrated into its genome, i.e., can be stably
transformed. Stably transformed cells typically retain the
introduced nucleic acid with each cell division. A plant or plant
cell can also be transiently transformed such that the construct is
not integrated into its genome. Transiently transformed cells
typically lose all or some portion of the introduced nucleic acid
construct with each cell division such that the introduced nucleic
acid cannot be detected in daughter cells after a sufficient number
of cell divisions. Both transiently transformed and stably
transformed transgenic plants and plant cells can be useful in the
methods described herein.
[0275] Transgenic plant cells used in methods described herein can
constitute part or all of a whole plant. Such plants can be grown
in a manner suitable for the species under consideration, either in
a growth chamber, a greenhouse, or in a field. Transgenic plants
can be bred as desired for a particular purpose, e.g., to introduce
a recombinant nucleic acid into other lines, to transfer a
recombinant nucleic acid to other species, or for further selection
of other desirable traits. Alternatively, transgenic plants can be
propagated vegetatively for those species amenable to such
techniques. As used herein, a transgenic plant also refers to
progeny of an initial transgenic plant. Progeny includes
descendants of a particular plant or plant line. Progeny of an
instant plant include seeds formed on F.sub.1, F.sub.2, F.sub.3,
F.sub.4, F.sub.5, F.sub.6 and subsequent generation plants, or
seeds formed on BC.sub.1, BC.sub.2, BC.sub.3, and subsequent
generation plants, or seeds formed on F.sub.1BC.sub.1,
F.sub.1BC.sub.2, F.sub.1BC.sub.3, and subsequent generation plants.
The designation F.sub.1 refers to the progeny of a cross between
two parents that are genetically distinct. The designations
F.sub.2, F.sub.3, F.sub.4, F.sub.5 and F.sub.6 refer to subsequent
generations of self- or sib-pollinated progeny of an F.sub.1 plant.
Seeds produced by a transgenic plant can be grown and then selfed
(or outcrossed and selfed) to obtain seeds homozygous for the
nucleic acid construct.
[0276] Transgenic plants can be grown in suspension culture, or
tissue or organ culture. For the purposes of this invention, solid
and/or liquid tissue culture techniques can be used. When using
solid medium, transgenic plant cells can be placed directly onto
the medium or can be placed onto a filter that is then placed in
contact with the medium. When using liquid medium, transgenic plant
cells can be placed onto a flotation device, e.g., a porous
membrane that contacts the liquid medium. Solid medium typically is
made from liquid medium by adding agar. For example, a solid medium
can be Murashige and Skoog (MS) medium containing agar and a
suitable concentration of an auxin, e.g., 2,4-dichlorophenoxyacetic
acid (2,4-D), and a suitable concentration of a cytokinin, e.g.,
kinetin.
[0277] When transiently transformed plant cells are used, a
reporter sequence encoding a reporter polypeptide having a reporter
activity can be included in the transformation procedure and an
assay for reporter activity or expression can be performed at a
suitable time after transformation. A suitable time for conducting
the assay typically is about 1-21 days after transformation, e.g.,
about 1-14 days, about 1-7 days, or about 1-3 days. The use of
transient assays is particularly convenient for rapid analysis in
different species, or to confirm expression of a heterologous
oil-modulating polypeptide whose expression has not previously been
confirmed in particular recipient cells.
[0278] Techniques for introducing nucleic acids into
monocotyledonous and dicotyledonous plants are known in the art,
and include, without limitation, Agrobacterium-mediated
transformation, viral vector-mediated transformation,
electroporation and particle gun transformation, e.g., U.S. Pat.
Nos. 5,538,880; 5,204,253; 6,329,571 and 6,013,863. If a cell or
cultured tissue is used as the recipient tissue for transformation,
plants can be regenerated from transformed cultures if desired, by
techniques known to those skilled in the art.
[0279] In aspects related to making transgenic plants, a typical
step involves selection or screening of transformed plants, e.g.,
for the presence of a functional vector as evidenced by expression
of a selectable marker. Selection or screening can be carried out
among a population of recipient cells to identify transformants
using selectable marker genes such as herbicide resistance genes.
Physical and biochemical methods can be used to identify
transformants. These include Southern analysis or PCR amplification
for detection of a polynucleotide; Northern blots, S1 RNase
protection, primer-extension, or RT-PCR amplification for detecting
RNA transcripts; enzymatic assays for detecting enzyme or ribozyme
activity of polypeptides and polynucleotides; and protein gel
electrophoresis, Western blots, immunoprecipitation, and
enzyme-linked immunoassays to detect polypeptides. Other techniques
such as in situ hybridization, enzyme staining, and immunostaining
also can be used to detect the presence or expression of
polypeptides and/or polynucleotides. Methods for performing all of
the referenced techniques are known.
[0280] A population of transgenic plants can be screened and/or
selected for those members of the population that have a desired
trait or phenotype conferred by expression of the transgene. For
example, a population of progeny of a single transformation event
can be screened for those plants having a desired level of
expression of a heterologous oil-modulating polypeptide or nucleic
acid. As an alternative, a population of plants comprising
independent transformation events can be screened for those plants
having a desired trait, such as a modulated level of oil. Selection
and/or screening can be carried out over one or more generations,
which can be useful to identify those plants that have a
statistically significant difference in an oil level as compared to
a corresponding level in a control plant. Selection and/or
screening can also be carried out in more than one geographic
location. In some cases, transgenic plants can be grown and
selected under conditions which induce a desired phenotype or are
otherwise necessary to produce a desired phenotype in a transgenic
plant. In addition, selection and/or screening can be carried out
during a particular developmental stage in which the phenotype is
expected to be exhibited by the plant. Selection and/or screening
can be carried out to choose those transgenic plants having a
statistically significant difference in an oil level relative to a
control plant that lacks the transgene. Selected or screened
transgenic plants have an altered phenotype as compared to a
corresponding control plant, as described in the "Transgenic Plant
Phenotypes" section below.
Plant Species
[0281] The polynucleotides and vectors described herein can be used
to transform a number of monocotyledonous and dicotyledonous plants
and plant cell systems, including dicots such as alfalfa, almond,
amaranth, apple, apricot, avocado, beans (including kidney beans,
lima beans, dry beans, green beans), broccoli, cabbage, canola,
carrot, cashew, castor bean, cherry, chick peas, chicory, clover,
cocoa, coffee, cotton, crambe, flax, grape, grapefruit, hazelnut,
hemp, jatropha, jojoba, lemon, lentils, lettuce, linseed, mango,
melon (e.g., watermelon, cantaloupe), mustard, neem, olive, orange,
peach, peanut, pear, peas, pepper, plum, poppy, potato, pumpkin,
oilseed rape, rapeseed (high erucic acid and canola), safflower,
sesame, soybean, spinach, strawberry, sugar beet, sunflower, sweet
potatoes, tea, tomato, walnut, and yams, as well as monocots such
as banana, barley, bluegrass, coconut, date palm, fescue, field
corn, garlic, millet, oat, oil palm, onion, palm kernel oil,
pineapple, popcorn, rice, rye, ryegrass, sorghum, sudangrass,
sugarcane, sweet corn, switchgrass, timothy, and wheat. Brown
seaweeds, green seaweeds, red seaweeds, and microalgae can also be
used.
[0282] Thus, the methods and compositions described herein can be
used with dicotyledonous plants belonging, for example, to the
orders Apiales, Arecales, Aristochiales, Asterales, Batales,
Campanulales, Capparales, Caryophyllales, Casuarinales,
Celastrales, Cornales, Cucurbitales, Diapensales, Dilleniales,
Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales,
Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales,
Illiciales, Juglandales, Lamiales, Laurales, Lecythidales,
Leitneriales, Linales, Magniolales, Malvales, Myricales, Myrtales,
Nymphaeales, Papaverales, Piperales, Plantaginales, Plumbaginales,
Podostemales, Polemoniales, Polygalales, Polygonales, Primulales,
Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales,
Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae,
Scrophulariales, Solanales, Trochodendrales, Theales, Umbellales,
Urticates, and Violates. The methods and compositions described
herein also can be utilized with monocotyledonous plants such as
those belonging to the orders Alismatales, Arales, Arecales,
Asparagales, Bromeliales, Commelinales, Cyclanthales, Cyperales,
Eriocaulales, Hydrocharitales, Juncales, Liliales, Najadales,
Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales,
Zingiberales, and with plants belonging to Gymnospermae, e.g.,
Cycadales, Ginkgoales, Gnetales, and Pinales.
[0283] The methods and compositions can be used over a broad range
of plant species, including species from the dicot genera
Amaranthus, Anacardium, Arachis, Azadirachta, Brassica, Calendula,
Camellia, Canarium, Cannabis, Capsicum, Carthamus, Cicer,
Cichorium, Cinnamomum, Citrus, Citrullus, Coffea, Corylus, Crambe,
Cucumis, Cucurbita, Daucus, Dioscorea, Fragaria, Glycine,
Gossypium, Helianthus, Jatropha, Juglans, Lactuca, Lens, Linum,
Lycopersicon, Malus, Mangifera, Medicago, Mentha, Nicotiana,
Ocimum, Olea, Papaver, Persea, Phaseolus, Pistacia, Pisum, Prunus,
Pyrus, Ricinus, Rosmarinus, Salvia, Sesamum, Simmondsia, Solanum,
Spinacia, Theobroma, Thymus, Trifolium, Vaccinium, Vigna, and
Vitis; and the monocot genera Allium, Ananas, Asparagus, Avena,
Cocos, Curcuma, Elaeis, Festuca, Festulolium, Hordeum, Lemna,
Lolium, Musa, Oryza, Panicum, Pennisetum, Phleum, Poa, Saccharum,
Secale, Sorghum, Triticosecale, Triticum, and Zea; and the
gymnosperm genera Abies, Cunninghamia, Picea, Pinus, Populus, and
Pseudotsuga.
[0284] The methods and compositions described herein also can be
used with brown seaweeds, e.g., Ascophyllum nodosum, Fucus
vesiculosus, Fucus serratus, Himanthalia elongata, and Undaria
pinnatifida; red seaweeds, e.g., Chondrus crispus, Cracilaria
verrucosa, Porphyra umbilicalis, and Palmaria palmata; green
seaweeds, e.g., Enteromorpha spp. and Ulva spp.; and microalgae,
e.g., Spirulina spp. (S. platensis and S. maxima) and Odontella
aurita. In addition, the methods and compositions can be used with
Crypthecodinium cohnii, Schizochytrium spp., and Haematococcus
pluvialis.
[0285] In some embodiments, a plant is a member of the species
Arachis hypogea, Brassica spp., Carthamus tinctorius, Elaeis
oleifera, Glycine max, Gossypium spp., Helianthus annuus, Jatropha
curcas, Linum usitatissimum, Triticum aestivum, or Zea mays.
Expression of Oil-Modulating Polypeptides
[0286] The polynucleotides and recombinant vectors described herein
can be used to express an oil-modulating polypeptide in a plant
species of interest. The term "expression" refers to the process of
converting genetic information of a polynucleotide into RNA through
transcription, which is catalyzed by an enzyme, RNA polymerase, and
into protein, through translation of mRNA on ribosomes.
"Up-regulation" or "activation" refers to regulation that increases
the production of expression products (mRNA, polypeptide, or both)
relative to basal or native states, while "down-regulation" or
"repression" refers to regulation that decreases production of
expression products (mRNA, polypeptide, or both) relative to basal
or native states.
[0287] The polynucleotides and recombinant vectors described herein
can be used to inhibit expression of an oil-modulating polypeptide
in a plant species of interest. A number of nucleic acid based
methods, including antisense RNA, ribozyme directed RNA cleavage,
post-transcriptional gene silencing (PTGS), e.g., RNA interference
(RNAi), and transcriptional gene silencing (TGS) can be used to
inhibit gene expression in plants. Antisense technology is one
well-known method. In this method, a nucleic acid segment from a
gene to be repressed is cloned and operably linked to a regulatory
region and a transcription termination sequence so that the
antisense strand of RNA is transcribed. The recombinant vector is
then transformed into plants, as described herein, and the
antisense strand of RNA is produced. The nucleic acid segment need
not be the entire sequence of the gene to be repressed, but
typically will be substantially complementary to at least a portion
of the sense strand of the gene to be repressed. Generally, higher
homology can be used to compensate for the use of a shorter
sequence. Typically, a sequence of at least 30 nucleotides is used,
e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more.
[0288] In another method, a nucleic acid can be transcribed into a
ribozyme, or catalytic RNA, that affects expression of an mRNA.
See, U.S. Pat. No. 6,423,885. Ribozymes can be designed to
specifically pair with virtually any target RNA and cleave the
phosphodiester backbone at a specific location, thereby
functionally inactivating the target RNA. Heterologous nucleic
acids can encode ribozymes designed to cleave particular mRNA
transcripts, thus preventing expression of a polypeptide.
Hammerhead ribozymes are useful for destroying particular mRNAs,
although various ribozymes that cleave mRNA at site-specific
recognition sequences can be used. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target RNA contain a 5'-UG-3' nucleotide sequence. The
construction and production of hammerhead ribozymes is known in the
art. See, for example, U.S. Pat. No. 5,254,678 and WO 02/46449 and
references cited therein. Hammerhead ribozyme sequences can be
embedded in a stable RNA such as a transfer RNA (tRNA) to increase
cleavage efficiency in vivo. Perriman et al., Proc. Natl. Acad.
Sci. USA, 92(13):6175-6179 (1995); de Feyter and Gaudron, Methods
in Molecular Biology, Vol. 74, Chapter 43, "Expressing Ribozymes in
Plants", Edited by Turner, P. C., Humana Press Inc., Totowa, N.J.
RNA endoribonucleases which have been described, such as the one
that occurs naturally in Tetrahymena thermophila, can be useful.
See, for example, U.S. Pat. Nos. 4,987,071 and 6,423,885.
[0289] PTGS, e.g., RNAi, can also be used to inhibit the expression
of a gene. For example, a construct can be prepared that includes a
sequence that is transcribed into an RNA that can anneal to itself,
e.g., a double stranded RNA having a stem-loop structure. In some
embodiments, one strand of the stem portion of a double stranded
RNA comprises a sequence that is similar or identical to the sense
coding sequence of an oil-modulating polypeptide, and that is from
about 10 nucleotides to about 2,500 nucleotides in length. The
length of the sequence that is similar or identical to the sense
coding sequence can be from 10 nucleotides to 500 nucleotides, from
15 nucleotides to 300 nucleotides, from 20 nucleotides to 100
nucleotides, or from 25 nucleotides to 100 nucleotides. The other
strand of the stem portion of a double stranded RNA comprises a
sequence that is similar or identical to the antisense strand of
the coding sequence of the oil-modulating polypeptide, and can have
a length that is shorter, the same as, or longer than the
corresponding length of the sense sequence. In some cases, one
strand of the stem portion of a double stranded RNA comprises a
sequence that is similar or identical to the 3' or 5' untranslated
region of an mRNA encoding an oil-modulating polypeptide, and the
other strand of the stem portion of the double stranded RNA
comprises a sequence that is similar or identical to the sequence
that is complementary to the 3' or 5' untranslated region,
respectively, of the mRNA encoding the oil-modulating polypeptide.
In other embodiments, one strand of the stem portion of a double
stranded RNA comprises a sequence that is similar or identical to
the sequence of an intron in the pre-mRNA encoding an
oil-modulating polypeptide, and the other strand of the stem
portion comprises a sequence that is similar or identical to the
sequence that is complementary to the sequence of the intron in the
pre-mRNA. The loop portion of a double stranded RNA can be from 3
nucleotides to 5,000 nucleotides, e.g., from 3 nucleotides to 25
nucleotides, from 15 nucleotides to 1,000 nucleotides, from 20
nucleotides to 500 nucleotides, or from 25 nucleotides to 200
nucleotides. The loop portion of the RNA can include an intron. A
double stranded RNA can have zero, one, two, three, four, five,
six, seven, eight, nine, ten, or more stem-loop structures. A
construct including a sequence that is operably linked to a
regulatory region and a transcription termination sequence, and
that is transcribed into an RNA that can form a double stranded
RNA, is transformed into plants as described herein. Methods for
using RNAi to inhibit the expression of a gene are known to those
of skill in the art. See, e.g., U.S. Pat. Nos. 5,034,323;
6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588. See also
WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S. Patent
Publications 20030175965, 20030175783, 20040214330, and
20030180945.
[0290] Constructs containing regulatory regions operably linked to
nucleic acid molecules in sense orientation can also be used to
inhibit the expression of a gene. The transcription product can be
similar or identical to the sense coding sequence of an
oil-modulating polypeptide. The transcription product can also be
unpolyadenylated, lack a 5' cap structure, or contain an
unsplicable intron. Methods of inhibiting gene expression using a
full-length cDNA as well as a partial cDNA sequence are known in
the art. See, e.g., U.S. Pat. No. 5,231,020.
[0291] In some embodiments, a construct containing a nucleic acid
having at least one strand that is a template for both sense and
antisense sequences that are complementary to each other is used to
inhibit the expression of a gene. The sense and antisense sequences
can be part of a larger nucleic acid molecule or can be part of
separate nucleic acid molecules having sequences that are not
complementary. The sense or antisense sequence can be a sequence
that is identical or complementary to the sequence of an mRNA, the
3' or 5' untranslated region of an mRNA, or an intron in a pre-mRNA
encoding an oil-modulating polypeptide. In some embodiments, the
sense or antisense sequence is identical or complementary to a
sequence of the regulatory region that drives transcription of the
gene encoding an oil-modulating polypeptide. In each case, the
sense sequence is the sequence that is complementary to the
antisense sequence.
[0292] The sense and antisense sequences can be any length greater
than about 12 nucleotides (e.g., 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides). For
example, an antisense sequence can be 21 or 22 nucleotides in
length. Typically, the sense and antisense sequences range in
length from about 15 nucleotides to about 30 nucleotides, e.g.,
from about 18 nucleotides to about 28 nucleotides, or from about 21
nucleotides to about 25 nucleotides.
[0293] In some embodiments, an antisense sequence is a sequence
complementary to an mRNA sequence encoding an oil-modulating
polypeptide described herein. The sense sequence complementary to
the antisense sequence can be a sequence present within the mRNA of
the oil-modulating polypeptide. Typically, sense and antisense
sequences are designed to correspond to a 15-30 nucleotide sequence
of a target mRNA such that the level of that target mRNA is
reduced.
[0294] In some embodiments, a construct containing a nucleic acid
having at least one strand that is a template for more than one
sense sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sense
sequences) can be used to inhibit the expression of a gene.
Likewise, a construct containing a nucleic acid having at least one
strand that is a template for more than one antisense sequence
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more antisense sequences) can be
used to inhibit the expression of a gene. For example, a construct
can contain a nucleic acid having at least one strand that is a
template for two sense sequences and two antisense sequences. The
multiple sense sequences can be identical or different, and the
multiple antisense sequences can be identical or different. For
example, a construct can have a nucleic acid having one strand that
is a template for two identical sense sequences and two identical
antisense sequences that are complementary to the two identical
sense sequences. Alternatively, an isolated nucleic acid can have
one strand that is a template for (1) two identical sense sequences
20 nucleotides in length, (2) one antisense sequence that is
complementary to the two identical sense sequences 20 nucleotides
in length, (3) a sense sequence 30 nucleotides in length, and (4)
three identical antisense sequences that are complementary to the
sense sequence 30 nucleotides in length. The constructs provided
herein can be designed to have any arrangement of sense and
antisense sequences. For example, two identical sense sequences can
be followed by two identical antisense sequences or can be
positioned between two identical antisense sequences.
[0295] A nucleic acid having at least one strand that is a template
for one or more sense and/or antisense sequences can be operably
linked to a regulatory region to drive transcription of an RNA
molecule containing the sense and/or antisense sequence(s). In
addition, such a nucleic acid can be operably linked to a
transcription terminator sequence, such as the terminator of the
nopaline synthase (nos) gene. In some cases, two regulatory regions
can direct transcription of two transcripts: one from the top
strand, and one from the bottom strand. See, for example, Yan et
al., Plant Physiol., 141:1508-1518 (2006). The two regulatory
regions can be the same or different. The two transcripts can form
double-stranded RNA molecules that induce degradation of the target
RNA. In some cases, a nucleic acid can be positioned within a T-DNA
or P-DNA such that the left and right T-DNA border sequences, or
the left and right border-like sequences of the P-DNA, flank or are
on either side of the nucleic acid. The nucleic acid sequence
between the two regulatory regions can be from about 15 to about
300 nucleotides in length. In some embodiments, the nucleic acid
sequence between the two regulatory regions is from about 15 to
about 200 nucleotides in length, from about 15 to about 100
nucleotides in length, from about 15 to about 50 nucleotides in
length, from about 18 to about 50 nucleotides in length, from about
18 to about 40 nucleotides in length, from about 18 to about 30
nucleotides in length, or from about 18 to about 25 nucleotides in
length.
[0296] In some nucleic-acid based methods for inhibition of gene
expression in plants, a suitable nucleic acid can be a nucleic acid
analog. Nucleic acid analogs can be modified at the base moiety,
sugar moiety, or phosphate backbone to improve, for example,
stability, hybridization, or solubility of the nucleic acid.
Modifications at the base moiety include deoxyuridine for
deoxythymidine, and 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the
sugar moiety include modification of the 2' hydroxyl of the ribose
sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose
phosphate backbone can be modified to produce morpholino nucleic
acids, in which each base moiety is linked to a six-membered
morpholino ring, or peptide nucleic acids, in which the
deoxyphosphate backbone is replaced by a pseudopeptide backbone and
the four bases are retained. See, for example, Summerton and
Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et
al., Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the
deoxyphosphate backbone can be replaced with, for example, a
phosphorothioate or phosphorodithioate backbone, a
phosphoroamidite, or an alkyl phosphotriester backbone.
Transgenic Plant Phenotypes
[0297] In some embodiments, a plant in which expression of an
oil-modulating polypeptide is modulated can have increased levels
of seed oil. For example, an oil-modulating polypeptide described
herein can be expressed in a transgenic plant, resulting in
increased levels of seed oil. The seed oil level can be increased
by at least 2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, or more than 75 percent, as compared to the seed oil level
in a corresponding control plant that does not express the
transgene. In some embodiments, a plant in which expression of an
oil-modulating polypeptide is modulated can have decreased levels
of seed oil. The seed oil level can be decreased by at least 2
percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35
percent, as compared to the seed oil level in a corresponding
control plant that does not express the transgene.
[0298] Plants for which modulation of levels of seed oil can be
useful include, without limitation, almond, cashew, castor bean,
coconut, corn, cotton, flax, hazelnut, hemp, jatropha, linseed,
mustard, neem, oil palm, peanut, poppy, pumpkin, rapeseed, rice,
safflower, sesame seed, soybean, sunflower, and walnut. Increases
in seed oil in such plants can provide increased yields of oil
extracted from the seed and increased caloric content in foodstuffs
and animal feed produced from the seed. Decreases in seed oil in
such plants can be useful in situations where caloric intake should
be restricted.
[0299] In some embodiments, a plant in which expression of an
oil-modulating polypeptide is modulated can have increased or
decreased levels of oil in one or more non-seed tissues, e.g., leaf
tissues, stem tissues, root or corm tissues, or fruit tissues other
than seed. For example, the oil level can be increased by at least
2 percent, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or
more than 75 percent, as compared to the oil level in a
corresponding control plant that does not express the transgene. In
some embodiments, a plant in which expression of an oil-modulating
polypeptide is modulated can have decreased levels of oil in one or
more non-seed tissues. The oil level can be decreased by at least 2
percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or more than 35
percent, as compared to the oil level in a corresponding control
plant that does not express the transgene.
[0300] Plants for which modulation of levels of oil in non-seed
tissues can be useful include, without limitation, alfalfa, apple,
avocado, beans, carrot, cherry, coconut, coffee, grapefruit, lemon,
lettuce, oat, olive, onion, orange, palm, peach, peanut, pear,
pineapple, potato, ryegrass, sudangrass, switchgrass, and tomato.
Increases in non-seed oil in such plants can provide increased oil
and caloric content in edible plants, including animal forage.
[0301] In some embodiments, a plant in which expression of an
oil-modulating polypeptide having an amino acid sequence
corresponding to SEQ ID NO:94, SEQ ID NO:81, SEQ ID NO:111, or SEQ
ID NO:136 is modulated can have increased levels of seed protein
accompanying increased levels of seed oil. The protein level can be
increased by at least 2 percent, e.g., 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, or 40 percent, as compared to the protein level in a
corresponding control plant that does not express the
transgene.
[0302] Typically, a difference (e.g., an increase) in the amount of
oil or protein in a transgenic plant or cell relative to a control
plant or cell is considered statistically significant at p<0.05
with an appropriate parametric or non-parametric statistic, e.g.,
Chi-square test, Student's t-test, Mann-Whitney test, or F-test. In
some embodiments, a difference in the amount of oil or protein is
statistically significant at p<0.01, p<0.005, or p<0.001.
A statistically significant difference in, for example, the amount
of oil in a transgenic plant compared to the amount in cells of a
control plant indicates that the recombinant nucleic acid present
in the transgenic plant results in altered oil levels.
[0303] The phenotype of a transgenic plant is evaluated relative to
a control plant that does not express the exogenous polynucleotide
of interest, such as a corresponding wild type plant, a
corresponding plant that is not transgenic for the exogenous
polynucleotide of interest but otherwise is of the same genetic
background as the transgenic plant of interest, or a corresponding
plant of the same genetic background in which expression of the
polypeptide is suppressed, inhibited, or not induced (e.g., where
expression is under the control of an inducible promoter). A plant
is said "not to express" a polypeptide when the plant exhibits less
than 10%, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,
0.1%, 0.01%, or 0.001%, of the amount of polypeptide or mRNA
encoding the polypeptide exhibited by the plant of interest.
Expression can be evaluated using methods including, for example,
RT-PCR, Northern blots, S1 RNase protection, primer extensions,
Western blots, protein gel electrophoresis, immunoprecipitation,
enzyme-linked immunoassays, chip assays, and mass spectrometry. It
should be noted that if a polypeptide is expressed under the
control of a tissue-preferential or broadly expressing promoter,
expression can be evaluated in the entire plant or in a selected
tissue. Similarly, if a polypeptide is expressed at a particular
time, e.g., at a particular time in development or upon induction,
expression can be evaluated selectively at a desired time
period.
[0304] Information that the polypeptides disclosed herein can
modulate oil content can be useful in breeding of crop plants.
Based on the effect of disclosed polypeptides on oil content, one
can search for and identify polymorphisms linked to genetic loci
for such polypeptides. Polymorphisms that can be identified include
simple sequence repeats (SSRs), rapid amplification of polymorphic
DNA (RAPDs), amplified fragment length polymorphisms (AFLPs) and
restriction fragment length polymorphisms (RFLPs).
[0305] If a polymorphism is identified, its presence and frequency
in populations is analyzed to determine if it is statistically
significantly correlated to an alteration in oil content. Those
polymorphisms that are correlated with an alteration in oil content
can be incorporated into a marker assisted breeding program to
facilitate the development of lines that have a desired alteration
in oil content. Typically, a polymorphism identified in such a
manner is used with polymorphisms at other loci that are also
correlated with a desired alteration in oil content.
Articles of Manufacture
[0306] Transgenic plants provided herein have particular uses in
the agricultural and nutritional industries. For example,
transgenic plants described herein can be used to make food
products and animal feed. Suitable plants with which to make such
products include almond, avocado, cashew, coconut, corn, flax,
olive, peanut, soybean, sunflower, and walnut. Such products are
useful to provide desired oil and caloric content in the diet.
[0307] Transgenic plants provided herein can also be used to make
vegetable oil. Vegetable oils can be chemically extracted from
transgenic plants using a solvent, such as hexane. In some cases,
olive, coconut and palm oils can be produced by mechanical
extraction, such as expeller-pressed extraction. Oil presses, such
as the screw press and the ram press, can also be used. Suitable
plants from which to make oil include almond, apricot, avocado,
canola, cashew, castor bean, coconut, corn, cotton, flax, grape,
hazelnut, hemp, mustard, neem, olive, palm, peanut, poppy, pumpkin,
rapeseed, rice, safflower, sesame, soybean, sunflower, and walnut.
Such oils can be used for frying, baking, and spray coating
applications. Vegetable oils also can be used to make margarine,
processed foods, oleochemicals, and essential oils. Vegetable oils
are used in the electrical industry as insulators. Vegetable oils
are also used as lubricants. Vegetable oil derivatives can be used
in the manufacture of polymers.
[0308] Vegetable oil from transgenic plants provided herein can
also be used as fuel. For example, vegetable oil can be used as
fuel in a vehicle that heats the oil before it enters the fuel
system. Heating vegetable oil to 150.degree. F. reduces the
viscosity of the oil sufficiently for use in diesel engines, such
as Mercedes-Benz.RTM. diesel engines. The viscosity of the oil can
also be reduced before it enters the tank so that neither the
engine or the vehicle needs modification. Methods of reducing oil
viscosity include: transesterification, pyrolysis, micro emulsion,
blending and thermal depolymerization. The transesterification
refining process creates esters from vegetable oil by using an
alcohol in the presence of a catalyst. This reaction takes a
triglyceride molecule, or a complex fatty acid, neutralizes the
free fatty acids and removes the glycerin, thereby creating an
alcohol ester. One method of transesterification mixes methanol
with sodium hydroxide and then aggressively mixes the resulting
methoxide with vegetable oil, which results in a methyl ester.
Ester-based oxygenated fuel made from vegetable oil is known as
biodiesel. Biodiesel can be used as a pure fuel or blended with
petroleum in any percentage. B5 biodiesel, for example, is a blend
of 5% biodiesel and 95% petroleum diesel. B20 biodiesel, including
BioWillie.RTM. diesel fuel, is produced by blending 20% biodiesel
and 80% petroleum diesel.
[0309] Use of biodiesel is beneficial for the environment because
it is associated with reduced emissions compared to the use of
petroleum diesel. In addition, biodiesel is a biodegradable,
nontoxic fuel that is made from renewable materials. Plants that
can be used as sources of oil for biodiesel production include
canola, cotton, flax, jatropha, oil palm, safflower, soybean, and
sunflower.
[0310] Seeds of transgenic plants described herein can be
conditioned and bagged in packaging material by means known in the
art to form an article of manufacture. Packaging material such as
paper and cloth are well known in the art. A package of seed can
have a label e.g., a tag or label secured to the packaging
material, a label printed on the packaging material, or a label
inserted within the package. The label can indicate that plants
grown from the seeds contained within the package can produce a
crop having an altered level of oil relative to corresponding
control plants.
[0311] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Transgenic Plants
[0312] The following symbols are used in the Examples: T.sub.1:
first generation transformant; T.sub.2: second generation, progeny
of self-pollinated T.sub.1 plants; T.sub.3: third generation,
progeny of self-pollinated T.sub.2 plants; T.sub.4: fourth
generation, progeny of self-pollinated T.sub.3 plants. Independent
transformations are referred to as events.
[0313] The following is a list of nucleic acids that were isolated
from Arabidopsis thaliana plants. Ceres Clone 25429 (At3g44590; SEQ
ID NO:93) is a cDNA clone that is predicted to encode a 113 amino
acid (SEQ ID NO:94) ribosomal polypeptide. Ceres Clone 41573
(At5g15140; SEQ ID NO:80) is a cDNA clone that is predicted to
encode a 298 amino acid (SEQ ID NO:81) aldose 1-epimerase
polypeptide. Ceres Clone 5750 (At2g27960; SEQ ID NO:110) is a cDNA
clone that is predicted to encode an 87 amino acid (SEQ ID NO:111)
cyclin-dependent kinase regulatory subunit polypeptide. Ceres Clone
121021 (SEQ ID NO:151) is a DNA clone that is predicted to encode a
199 amino acid (SEQ ID NO:152) polypeptide. Ceres Clone 158765 (SEQ
ID NO:158) is a DNA clone that is predicted to encode a 149 amino
acid (SEQ ID NO:159) polypeptide. Ceres Clone 16403 (SEQ ID NO:170)
is a DNA clone that is predicted to encode a 238 amino acid (SEQ ID
NO:171) PsbP polypeptide. Ceres Clone 19244 (SEQ ID NO:175) is a
DNA clone that is predicted to encode a 220 amino acid (SEQ ID
NO:176) AP2 domain-containing polypeptide. Ceres Clone 28635 (SEQ
ID NO:177) is a DNA clone that is predicted to encode a 410 amino
acid (SEQ ID NO:178) squalene/phytoene synthase polypeptide. Ceres
Clone 35698 (SEQ ID NO:192) is a DNA clone that is predicted to
encode a 109 amino acid (SEQ ID NO:193) ubiquitin-conjugating
enzyme polypeptide. Ceres Clone 36412 (SEQ ID NO:331) is a DNA
clone that is predicted to encode a 358 amino acid (SEQ ID NO:332)
polypeptide. Ceres Clone 368 (SEQ ID NO:341) is a DNA clone that is
predicted to encode a 52 amino acid (SEQ ID NO:342) polypeptide.
Ceres Clone 41046 (SEQ ID NO:343) is a DNA clone that is predicted
to encode a 220 amino acid (SEQ ID NO:344) cytochrome p450
polypeptide. Ceres Clone 4829 (SEQ ID NO:345) is a DNA clone that
is predicted to encode a 180 amino acid (SEQ ID NO:346) polypeptide
having a DUF662 domain. Ceres Clone 5426 (SEQ ID NO:358) is a DNA
clone that is predicted to encode a 143 amino acid (SEQ ID NO:359)
ribosomal L28e protein family polypeptide. Ceres Clone 7894 (SEQ ID
NO:373) is a DNA clone that is predicted to encode a 512 amino acid
(SEQ ID NO:374) Major Facilitator Superfamily transporter
polypeptide. Ceres Clone 8161 (SEQ ID NO:397) is a cDNA clone that
is predicted to encode a 218 amino acid (SEQ ID NO:398)
polypeptide.
[0314] Ceres Clone 218626 (SEQ ID NO:135) is a cDNA clone that was
isolated from Zea mays and that is predicted to encode a 454 amino
acid (SEQ ID NO:136) tryptophan/tyrosine permease family
polypeptide.
[0315] Each isolated nucleic acid described above was cloned into a
Ti plasmid vector, CRS 338, containing a phosphinothricin
acetyltransferase gene which confers Finale.TM. resistance to
transformed plants. Constructs were made using CRS 338 that
contained Ceres Clone 25429, Ceres Clone 41573, Ceres Clone 5750,
Ceres Clone 218626, Ceres Clone 121021, Ceres Clone 158765, Ceres
Clone 16403, Ceres Clone 19244, Ceres Clone 28635, Ceres Clone
35698, Ceres Clone 36412, Ceres Clone 368, Ceres Clone 41046, Ceres
Clone 4829, Ceres Clone 5426, Ceres Clone 7894, or Ceres Clone
8161, each operably linked to a CaMV 35S promoter. Wild-type
Arabidopsis thaliana ecotype Wassilewskija (Ws) plants were
transformed separately with each construct. The transformations
were performed essentially as described in Bechtold et al., C.R.
Acad. Sci. Paris, 316:1194-1199 (1993).
[0316] Transgenic Arabidopsis lines containing Ceres Clone 25429,
Ceres Clone
[0317] 41573, Ceres Clone 5750, Ceres Clone 218626, Ceres Clone
5426, Ceres Clone 35698, Ceres Clone 4829, Ceres Clone 28635, Ceres
Clone 16403, Ceres Clone 41046, Ceres Clone 19244, Ceres Clone
7894, Ceres Clone 158765, Ceres Clone 121021, Ceres Clone 36412,
Ceres Clone 8161, or Ceres Clone 368 were designated ME01597,
ME01720, ME01833, ME02065, ME01902, ME00072, ME00085, ME00147,
ME00896, ME00900, ME00913, ME01704, ME02505, ME02525, ME00902,
ME00914, or ME01754, respectively. The presence of each vector
containing a Ceres clone described above in the respective
transgenic Arabidopsis line transformed with the vector was
confirmed by Finale.TM. resistance, polymerase chain reaction (PCR)
amplification from green leaf tissue extract, and/or sequencing of
PCR products. As controls, wild-type Arabidopsis ecotype Ws plants
were transformed with the empty vector CRS 338.
[0318] The physical appearances of T.sub.1 plants from ten events
each of ME01597 and ME01720 were similar to those of corresponding
control plants. The physical appearances of T.sub.1 plants from
nine out of ten events of ME01833 were comparable to those of the
controls. Event -08 of ME01833 was taller and had reduced fertility
compared to control plants. Event -08 of ME02065 also had reduced
fertility compared to control plants. The physical appearances of
T.sub.1 plants from nine additional events of ME02065 were similar
to those of corresponding control plants.
Example 2
Analysis of Oil Content in Transgenic Arabidopsis Seeds
[0319] An analytical method based on Fourier transform
near-infrared (FT-NIR) spectroscopy was developed, validated, and
used to perform a high-throughput screen of transgenic seed lines
for alterations in seed oil content. To calibrate the FT-NIR
spectroscopy method, a sub-population of transgenic seed lines was
randomly selected and analyzed for oil content using a direct
primary method. Fatty acid methyl ester (FAME) analysis by gas
chromatography-mass spectroscopy (GC-MS) was used as the direct
primary method to determine the total fatty acid content for each
seed line and produce the FT-NIR spectroscopy calibration curves
for oil.
[0320] To analyze seed oil content using GC-MS, seed tissue was
homogenized in liquid nitrogen using a mortar and pestle to create
a powder. The tissue was weighed, and 5.0.+-.0.25 mg were
transferred into a 2 mL Eppendorf tube. The exact weight of each
sample was recorded. One mL of 2.5% H.sub.2SO.sub.4 (v/v in
methanol) and 20 .mu.L of undecanoic acid internal standard (1
mg/mL in hexane) were added to the weighed seed tissue. The tubes
were incubated for two hours at 90.degree. C. in a pre-equilibrated
heating block. The samples were removed from the heating block and
allowed to cool to room temperature. The contents of each Eppendorf
tube were poured into a 15 mL polypropylene conical tube, and 1.5
mL of a 0.9% NaCl solution and 0.75 mL of hexane were added to each
tube. The tubes were vortexed for 30 seconds and incubated at room
temperature for 15 minutes. The samples were then centrifuged at
4,000 rpm for 5 minutes using a bench top centrifuge. If emulsions
remained, then the centrifugation step was repeated until they were
dissipated. One hundred .mu.L of the hexane (top) layer was
pipetted into a 1.5 mL autosampler vial with minimum volume insert.
The samples were stored no longer than 1 week at -80.degree. C.
until they were analyzed.
[0321] Samples were analyzed using a Shimadzu QP-2010 GC-MS
(Shimadzu Scientific Instruments, Columbia, Md.). The first and
last sample of each batch consisted of a blank (hexane). Every
fifth sample in the batch also consisted of a blank. Prior to
sample analysis, a 7-point calibration curve was generated using
the Supelco 37 component FAME mix (0.00004 mg/mL to 0.2 mg/mL). The
injection volume was 1 .mu.L.
[0322] The GC parameters were as follows: column oven temperature:
70.degree. C., inject temperature: 230.degree. C., inject mode:
split, flow control mode: linear velocity, column flow: 1.0 mL/min,
pressure: 53.5 mL/min, total flow: 29.0 mL/min, purge flow: 3.0
mL/min, split ratio: 25.0. The temperature gradient was as follows:
70.degree. C. for 5 minutes, increasing to 350.degree. C. at a rate
of 5 degrees per minute, and then held at 350.degree. C. for 1
minute. The MS parameters were as follows: ion source temperature:
200.degree. C., interface temperature: 240.degree. C., solvent cut
time: 2 minutes, detector gain mode: relative, detector gain: 0.6
kV, threshold: 1000, group: 1, start time: 3 minutes, end time: 62
minutes, ACQ mode: scan, interval: 0.5 second, scan speed: 666
amu/sec., start M/z: 40, end M/z: 350. The instrument was tuned
each time the column was cut or a new column was used.
[0323] The data were analyzed using the Shimadzu GC-MS Solutions
software. Peak areas were integrated and exported to an Excel
spreadsheet. Fatty acid peak areas were normalized to the internal
standard, the amount of tissue weighed, and the slope of the
corresponding calibration curve generated using the FAME mixture.
Peak areas were also multiplied by the volume of hexane (0.75 mL)
used to extract the fatty acids.
[0324] The same seed lines that were analyzed using GC-MS were also
analyzed by FT-NIR spectroscopy, and the oil values determined by
the GC-MS primary method were entered into the FT-NIR chemometrics
software (Bruker Optics, Billerica, Mass.) to create a calibration
curve for oil content. The actual oil content of each seed line
analyzed using GC-MS was plotted on the x-axis of the calibration
curve. The y-axis of the calibration curve represented the
predicted values based on the best-fit line. Data points were
continually added to the calibration curve data set.
[0325] T.sub.2 seed from each transgenic plant line was analyzed by
FT-NIR spectroscopy. Sarstedt tubes containing seeds were placed
directly on the lamp, and spectra were acquired through the bottom
of the tube. The spectra were analyzed to determine seed oil
content using the FT-NIR chemometrics software (Bruker Optics) and
the oil calibration curve. Results for experimental samples were
compared to population means and standard deviations calculated for
transgenic seed lines that were planted within 30 days of the lines
being analyzed and grown under the same conditions. Typically,
results from three to four events of each of 400 to 1600 different
transgenic lines were used to calculate a population mean. Each
data point was assigned a z-score (z=(x-mean)/std), and a p-value
was calculated for the z-score.
[0326] Transgenic seed lines with oil levels in T.sub.2 seed that
differed by more than two standard deviations from the population
mean were selected for evaluation of oil levels in the T.sub.3
generation. All events of selected lines were planted in individual
pots. The pots were arranged randomly in flats along with pots
containing matched control plants in order to minimize
microenvironment effects. Matched control plants contained an empty
version of the vector used to generate the transgenic seed lines.
T.sub.3 seed from up to five plants from each event was collected
and analyzed individually using FT-NIR spectroscopy. Data from
replicate samples were averaged and compared to controls using the
Student's t-test.
Example 3
Analysis of Protein Content in Transgenic Arabidopsis Seeds
[0327] An analytical method based on Fourier transform
near-infrared (FT-NIR) spectroscopy was developed, validated, and
used to perform a high-throughput screen of transgenic seed lines
for alterations in seed protein content. To calibrate the FT-NIR
spectroscopy method, total nitrogen elemental analysis was used as
a primary method to analyze a sub-population of randomly selected
transgenic seed lines. The overall percentage of nitrogen in each
sample was determined Percent nitrogen values were multiplied by a
conversion factor to obtain percent total protein values. A
conversion factor of 5.30 was selected based on data for cotton,
sunflower, safflower, and sesame seed (Rhee, K. C., Determination
of Total Nitrogen In Handbook of Food Analytical Chemistry--Water,
Proteins, Enzymes, Lipids, and Carbohydrates (R. Wrolstad et al.,
ed.), John Wiley and Sons, Inc., p. 105, (2005)). The same seed
lines were then analyzed by FT-NIR spectroscopy, and the protein
values calculated via the primary method were entered into the
FT-NIR chemometrics software (Bruker Optics, Billerica, Mass.) to
create a calibration curve for analysis of seed protein content by
FT-NIR spectroscopy.
[0328] Elemental analysis was performed using a FlashEA 1112 NC
Analyzer (Thermo Finnigan, San Jose, Calif.). To analyze total
nitrogen content, 2.00.+-.0.15 mg of dried transgenic Arabidopsis
seed was weighed into a tared tin cup. The tin cup with the seed
was weighed, crushed, folded in half, and placed into an
autosampler slot on the FlashEA 1112 NC Analyzer (Thermo Finnigan).
Matched controls were prepared in a manner identical to the
experimental samples and spaced evenly throughout the batch. The
first three samples in every batch were a blank (empty tin cup), a
bypass, (approximately 5 mg of aspartic acid), and a standard
(5.00.+-.0.15 mg aspartic acid), respectively. Blanks were entered
between every 15 experimental samples. Each sample was analyzed in
triplicate.
[0329] The FlashEA 1112 NC Analyzer (Thermo Finnigan) instrument
parameters were as follows: left furnace 900.degree. C., right
furnace 840.degree. C., oven 50.degree. C., gas flow carrier 130
mL/min., and gas flow reference 100 mL/min. The data parameter LLOD
was 0.25 mg for the standard and different for other materials. The
data parameter LLOQ was 3.0 mg for the standard, 1.0 mg for seed
tissue, and different for other materials.
[0330] Quantification was performed using the Eager 300 software
(Thermo Finnigan). Replicate percent nitrogen measurements were
averaged and multiplied by a conversion factor of 5.30 to obtain
percent total protein values. For results to be considered valid,
the standard deviation between replicate samples was required to be
less than 10%. The percent nitrogen of the aspartic acid standard
was required to be within .+-.1.0% of the theoretical value. For a
run to be declared valid, the weight of the aspartic acid
(standard) was required to be between 4.85 and 5.15 mg, and the
blank(s) were required to have no recorded nitrogen content.
[0331] The same seed lines that were analyzed for elemental
nitrogen content were also analyzed by FT-NIR spectroscopy, and the
percent total protein values determined by elemental analysis were
entered into the FT-NIR chemometrics software (Bruker Optics,
Billerica, Mass.) to create a calibration curve for protein
content. The protein content of each seed line based on total
nitrogen elemental analysis was plotted on the x-axis of the
calibration curve. The y-axis of the calibration curve represented
the predicted values based on the best-fit line. Data points were
continually added to the calibration curve data set.
[0332] T.sub.2 seed from each transgenic plant line was analyzed by
FT-NIR spectroscopy. Sarstedt tubes containing seeds were placed
directly on the lamp, and spectra were acquired through the bottom
of the tube. The spectra were analyzed to determine seed protein
content using the FT-NIR chemometrics software (Bruker Optics) and
the protein calibration curve. Results for experimental samples
were compared to population means and standard deviations
calculated for transgenic seed lines that were planted within 30
days of the lines being analyzed and grown under the same
conditions. Typically, results from three to four events of each of
400 to 1600 different transgenic lines were used to calculate a
population mean. Each data point was assigned a z-score
(z=(x-mean)/std), and a p-value was calculated for the z-score.
[0333] Transgenic seed lines with oil levels in T.sub.2 seed that
differed by more than two standard deviations from the population
mean were also analyzed to determine protein levels in the T.sub.3
generation. Events of selected lines were planted in individual
pots. The pots were arranged randomly in flats along with pots
containing matched control plants in order to minimize
microenvironment effects. Matched control plants contained an empty
version of the vector used to generate the transgenic seed lines.
T.sub.3 seed from up to five plants from each event was collected
and analyzed individually using FT-NIR spectroscopy. Data from
replicate samples were averaged and compared to controls using the
Student's t-test.
Example 4
Results for ME01597 Events
[0334] T.sub.2 and T.sub.3 seed from three events and four events,
respectively, of ME01597 containing Ceres Clone 25429 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0335] The oil content in T.sub.2 seed from two events of ME01597
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME01597. As presented in Table 1, the oil content was increased to
123% and 124% in seed from events -03 and -06, respectively,
compared to the population mean.
TABLE-US-00001 TABLE 1 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME01597 events containing Ceres Clone 25429 Event
-01 Event -03 Event -06 Event -09 Control Oil 101 123 124 No data
100 .+-. 11* content (% control) in T.sub.2 seed p-value** 0.32
0.04 0.03 No data N/A Oil 106 .+-. 2 107 .+-. 4 107 .+-. 3 99 .+-.
4 100 .+-. 4 content (% control) in T.sub.3 seed p-value***
<0.01 0.05 <0.01 0.70 N/A No. of T.sub.2 5 4 4 5 31 plants
*Population mean of the oil content of seed from transgenic lines
planted within 30 days of ME01597. Variation is presented as the
standard error of the mean. **The p-values for T.sub.2 seed were
calculated using z-scores. ***The p-values for T.sub.3 seed were
calculated using a Student's t-test.
[0336] The oil content in T.sub.3 seed from three events of ME01597
was significantly increased compared to the oil content of
corresponding control seeds. As presented in Table 1, the oil
content was increased to 106% in seed from event -01 and to 107% in
seed from events -03 and -06 compared to the oil content in control
seed.
[0337] T.sub.2 and T.sub.3 seed from three events and four events,
respectively, of ME01597 containing Ceres Clone 25429 was also
analyzed for protein content using FT-NIR spectroscopy as described
in Example 3.
[0338] The protein content in T.sub.2 seed from ME01597 events was
not observed to differ significantly from the mean protein content
in seed from transgenic Arabidopsis lines planted within 30 days of
ME01597 (Table 2).
TABLE-US-00002 TABLE 2 Protein content (% control) in T.sub.2 and
T.sub.3 seed from ME01597 events containing Ceres Clone 25429 Event
-01 Event -03 Event -06 Event -09 Control Protein 98 98 93 No data
100 .+-. 9* content (% control) in T.sub.2 seed p-value** 0.40 0.29
0.27 No data N/A Protein 109 .+-. 2 110 .+-. 4 106 .+-. 7 110 .+-.
3 100 .+-. 5 content (% control) in T.sub.3 seed p-value***
<0.01 0.01 0.15 <0.01 N/A No. of T.sub.2 5 4 4 5 31 plants
*Population mean of the protein content in seed from transgenic
lines planted within 30 days of ME01597. Variation is presented as
the standard error of the mean. **The p-values for T.sub.2 seed
were calculated using z-scores. ***The p-values for T.sub.3 seed
were calculated using a Student's t-test.
[0339] The protein content in T.sub.3 seed from three events of
ME01597 was significantly increased compared to the protein content
in corresponding control seed. As presented in Table 2, the protein
content was increased to 109% in seed from event -01 and to 110% in
seed from events -03 and -09 compared to the protein content in
control seed.
[0340] There were no observable or statistically significant
differences between T.sub.2 ME01597 and control plants in
germination, onset of flowering, rosette area, fertility, and
general morphology/architecture.
Example 5
Results for ME01720 Events
[0341] T.sub.2 and T.sub.3 seed from five events of ME01720
containing Ceres Clone 41573 was analyzed for oil content using
FT-NIR spectroscopy as described in Example 2.
[0342] The oil content in T.sub.2 seed from three events of ME01720
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME01720. As presented in Table 3, the oil content was increased to
123% in seed from events -01 and -03 and to 130% in seed from event
-06 compared to the population mean.
TABLE-US-00003 TABLE 3 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME01720 events containing Ceres Clone 41573 Event
Event Event Event Event -01 -02 -03 -04 -06 Control Oil content 123
98 123 112 130 100 .+-. 11* (% control) in T.sub.2 seed p-value**
0.04 0.31 0.04 0.18 0.01 N/A Oil content 105 .+-. 3 100 .+-. 3 108
.+-. 1 99 .+-. 1 105 .+-. 2 100 .+-. 4 (% control) in T.sub.3 seed
p-value*** 0.01 1.00 <0.01 0.60 0.04 N/A No. of T.sub.2 5 5 4 3
3 31 plants *Population mean of the oil content in seed from
transgenic lines planted within 30 days of ME01720. Variation is
presented as the standard error of the mean. **The p-values for
T.sub.2 seed were calculated using z-scores. ***The p-values for
T.sub.3 seed were calculated using a Student's t-test.
[0343] The oil content in T.sub.3 seed from three events of ME01720
was significantly increased compared to the oil content in
corresponding control seeds. As presented in Table 3, the oil
content was increased to 105% in seed from events -01 and -06 and
to 108% in seed from event -03 compared to the oil content in
control seed.
[0344] T.sub.2 and T.sub.3 seed from five events of ME01720
containing Ceres Clone 41573 was also analyzed for total protein
content using FT-NIR spectroscopy as described in Example 3.
[0345] The protein content in T.sub.2 seed from ME01720 events was
not observed to differ significantly from the mean protein content
in seed from transgenic Arabidopsis lines planted within 30 days of
ME01720 (Table 4).
TABLE-US-00004 TABLE 4 Protein content (% control) in T.sub.2 and
T.sub.3 seed from ME01720 events containing Ceres Clone 41573 Event
Event Event Event Event -01 -02 -03 -04 -06 Control Protein 92 103
89 96 84 100 .+-. 8* content (% control) in T.sub.2 seed p-value**
0.24 0.40 0.15 0.36 0.06 N/A Protein 107 .+-. 2 114 .+-. 3 106 .+-.
5 107 .+-. 0 106 .+-. 2 100 .+-. 5 content (% control) in T.sub.3
seed p-value*** <0.01 <0.01 0.07 <0.01 0.01 N/A No. of
T.sub.2 5 5 4 3 3 31 plants *Population mean of the protein content
in seed from transgenic lines planted within 30 days of ME01720.
Variation is presented as the standard error of the mean. **The
p-values for T.sub.2 seed were calculated using z-scores. ***The
p-values for T.sub.3 seed were calculated using a Student's
t-test.
[0346] The protein content in T.sub.3 seed from four events of
ME01720 was significantly increased compared to the protein content
in corresponding control seed. As presented in Table 4, the protein
content was increased to 107%, 114%, 107%, and 106% in events -01,
-02, -04, and -06, respectively, compared to the protein content in
control seed.
[0347] There were no observable or statistically significant
differences between T.sub.2 ME01720 and control plants in
germination, onset of flowering, rosette area, and fertility. The
general morphology/architecture of the plants appeared wild-type in
all instances except for event -01, which had a small (<30%),
but statistically significant (p<0.05), change in plant
size.
Example 6
Results for ME01833 Events
[0348] T.sub.2 and T.sub.3 seed from four events of ME01833
containing Ceres Clone 5750 was analyzed for oil content using
FT-NIR spectroscopy as described in Example 2.
[0349] The oil content in T.sub.2 seed from four events of ME01833
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME01833. As presented in Table 5, the oil content was increased to
135%, 138%, 143%, and 172% in seed from events -01, -02, -03, and
-04, respectively, compared to the population mean.
TABLE-US-00005 TABLE 5 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME01833 events containing Ceres Clone 5750 Event
-01 Event -02 Event -03 Event -04 Control Oil 135 138 143 172 100
.+-. 11* content (% control) in T.sub.2 seed p-value** <0.01
<0.01 <0.01 <0.01 N/A Oil 95 .+-. 4 102 .+-. 4 105 .+-. 1
112 .+-. 1 100 .+-. 4 content (% control) in T.sub.3 seed
p-value*** 0.08 0.35 <0.01 <0.01 N/A No. of T.sub.2 4 4 3 4
31 plants *Population mean of the oil content in seed from
transgenic lines planted within 30 days of ME01833. Variation is
presented as the standard error of the mean. **The p-values for
T.sub.2 seed were calculated using z-scores. ***The p-values for
T.sub.3 seed were calculated using a Student's t-test.
[0350] The oil content in T.sub.3 seed from two events of ME01833
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 5, the oil
content was increased to 105% and 112% in seed from events -03 and
-04, respectively, compared to the oil content in control seed.
[0351] T.sub.2 and T.sub.3 seed from four events of ME01833
containing Ceres Clone 5750 was also analyzed for total protein
content using FT-NIR spectroscopy as described in Example 3.
[0352] The protein content in T.sub.2 seed from one event of
ME01833 was significantly increased compared to the mean protein
content in seed from transgenic Arabidopsis lines planted within 30
days of ME01833. As presented in Table 6, the protein content was
increased to 123% in event -04 compared to the population mean.
TABLE-US-00006 TABLE 6 Protein content (% control) in T.sub.2 and
T.sub.3 seed from ME01833 events containing Ceres Clone 5750 Event
-01 Event -02 Event -03 Event -04 Control Protein 94 101 98 123 100
.+-. 8* content (% control) in T.sub.2 seed p-value** 0.32 0.41
0.40 0.01 N/A Protein 109 .+-. 8 102 .+-. 3 99 .+-. 1 112 .+-. 5
100 .+-. 4 content (% control) in T.sub.3 seed p-value*** 0.11 0.28
0.28 0.02 N/A No. of T.sub.2 4 4 3 4 31 plants *Population mean of
the protein content in seed from transgenic lines planted within 30
days of ME01833. Variation is presented as the standard error of
the mean. **The p-values for T.sub.2 seed were calculated using
z-scores. ***The p-values for T.sub.3 seed were calculated using a
Student's t-test.
[0353] The protein content in T.sub.3 seed from one event of
ME01833 was significantly increased compared to the protein content
in corresponding control seed. As presented in Table 6, the protein
content was increased to 112% in seed from event -04 compared to
the protein content in control seed.
[0354] There were no observable or statistically significant
differences between T.sub.2 ME01833 and control plants in
germination, onset of flowering, rosette area, fertility, and
general morphology/architecture.
Example 7
Results for ME02065 Events
[0355] T.sub.2 and T.sub.3 seed from four events of ME02065
containing Ceres Clone
[0356] 218626 was analyzed for oil content using FT-NIR
spectroscopy as described in Example 2.
[0357] The oil content in T.sub.2 seed from three events of ME02065
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME02065. As presented in Table 7, the oil content was increased to
123%, 132%, and 145% in seed from events -01, -04, and -05,
respectively, compared to the population mean.
TABLE-US-00007 TABLE 7 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME02065 events containing Ceres Clone 218626
Event -01 Event -02 Event -04 Event -05 Control Oil 123 115 132 145
100 .+-. 11* content (% control) in T.sub.2 seed p-value** 0.04
0.13 <0.01 <0.01 N/A Oil 109 .+-. 4 103 .+-. 1 112 .+-. 1 102
.+-. 5 100 .+-. 4 content (% control) in T.sub.3 seed p-value***
0.01 0.05 <0.01 0.46 N/A No. of T.sub.2 4 2 5 4 31 plants
*Population mean of the oil content in seed from transgenic lines
planted within 30 days of ME02065. Variation is presented as the
standard error of the mean. **The p-values for T.sub.2 seed were
calculated using z-scores. ***The p-values for T.sub.3 seed were
calculated using a Student's t-test.
[0358] The oil content in T.sub.3 seed from three events of ME02065
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 7, the oil
content was increased to 109%, 103%, and 112% in seed from events
-01, -02, and -04, respectively, compared to the oil content in
control seed.
[0359] T.sub.2 and T.sub.3 seed from four events of ME02065
containing Ceres Clone 218626 was also analyzed for total protein
content using FT-NIR spectroscopy as described in Example 3.
[0360] The protein content in T.sub.2 seed from ME02065 events was
not observed to differ significantly from the mean protein content
in seed from transgenic Arabidopsis lines planted within 30 days of
ME02065 (Table 8).
TABLE-US-00008 TABLE 8 Protein content (% control) in T.sub.2 and
T.sub.3 seed from ME02065 events containing Ceres Clone 218626
Event -01 Event -02 Event -04 Event -05 Control Protein 103 104 104
105 100 .+-. 8* content (% control) in T.sub.2 seed p-value** 0.39
0.37 0.37 0.34 N/A Protein 105 .+-. 3 110 .+-. 1 109 .+-. 2 113
.+-. 4 100 .+-. 5 content (% control) in T.sub.3 seed p-value***
0.01 <0.01 <0.01 <0.01 N/A No. of T.sub.2 4 2 5 4 31
plants *Population mean of the protein content in seed from
transgenic lines planted within 30 days of ME02065. Variation is
presented as the standard error of the mean. **The p-values for
T.sub.2 seed were calculated using z-scores. ***The p-values for
T.sub.3 seed were calculated using a Student's t-test.
[0361] The protein content in T.sub.3 seed from four events of
ME02065 was significantly increased compared to the protein content
in corresponding control seed. As presented in Table 8, the protein
content was increased to 105%, 110%, 109%, and 113% in seed from
events -01, -02, -04, and -05, respectively, compared to the
protein content in control seed.
[0362] There were no observable or statistically significant
differences between T.sub.2 ME02065 and control plants in
germination, onset of flowering, rosette area, fertility, and
general morphology/architecture.
Example 8
Results for ME01902 Events
[0363] T.sub.2 and T.sub.3 seed from five events and four events,
respectively, of ME01902 containing Ceres Clone 5426 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0364] The oil content in T.sub.2 seed from three events of ME01902
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME01902. As presented in Table 9, the oil content was increased to
118%, 125%, and 124% in seed from events -02, -04, and -07,
respectively, compared to the population mean.
TABLE-US-00009 TABLE 9 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME01902 events containing Ceres Clone 5426 Event
Event Event Event Event -01 -02 -03 -04 -07 Control Oil content 102
118 107 125 124 100* (% control) in T.sub.2 seed p-value** 0.17
0.03 0.14 0.01 0.01 N/A Oil content No data 108 .+-. 4 103 .+-. 1
104 .+-. 4 109 .+-. 5 100 (% control) in T.sub.3 seed p-value*** No
data 0.02 <0.01 0.10 0.03 N/A *Population mean of the oil
content in seed from transgenic lines planted within 30 days of
ME01902. Variation is presented as the standard error of the mean.
**The p-values for T.sub.2 seed were calculated using z-scores.
***The p-values for T.sub.3 seed were calculated using a Student's
t-test.
[0365] The oil content in T.sub.3 seed from three events of ME01902
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 9, the oil
content was increased to 108%, 103%, and 109% in seed from events
-02, -03, and -07, respectively, compared to the oil content in
control seed.
[0366] T.sub.2 and T.sub.3 seed from five events and four events,
respectively, of ME01902 containing Ceres Clone 5426 was also
analyzed for total protein content using FT-NIR spectroscopy as
described in Example 3.
[0367] The protein content in T.sub.2 seed from ME01902 events was
not observed to differ significantly from the mean protein content
in seed from transgenic Arabidopsis lines planted within 30 days of
ME01902 (Table 10).
TABLE-US-00010 TABLE 10 Protein content (% control) in T.sub.2 and
T.sub.3 seed from ME01902 events containing Ceres Clone 5426 Event
Event Event Event Event -01 -02 -03 -04 -07 Control Protein content
99 106 90 93 96 100* (% control) in T.sub.2 seed p-value** 0.26
0.18 0.08 0.15 0.22 N/A Protein content No data 116 .+-. 4 113 118
.+-. 2 111 .+-. 1 100 .+-. 1 (% control) in T.sub.3 seed p-value***
No data 0.02 <0.01 <0.01 <0.01 N/A *Population mean of the
protein content in seed from transgenic lines planted within 30
days of ME01902. Variation is presented as the standard error of
the mean. **The p-values for T.sub.2 seed were calculated using
z-scores. ***The p-values for T.sub.3 seed were calculated using a
Student's t-test.
[0368] The protein content in T.sub.3 seed from four events of
ME01902 was significantly increased compared to the protein content
in corresponding control seed. As presented in Table 10, the
protein content was increased to 116%, 113%, 118%, and 111% in seed
from events -02, -03, -04, and -07, respectively, compared to the
protein content in control seed.
Example 9
Results for ME00072 Events
[0369] T.sub.2 and T.sub.3 seed from seven events and five events,
respectively, of ME00072 containing Ceres Clone 35698 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0370] The oil content in T.sub.2 seed from three events of ME00072
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME00072. As presented in Table 11, the oil content was increased to
115%, 121%, and 117% in seed from events -02, -03, and -06,
respectively, compared to the population mean.
TABLE-US-00011 TABLE 11 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME00072 events containing Ceres Clone 35698 Event
Event Event Event Event Event Event -02 -03 -05 -06 -07 -08 -09
Control Oil content (% 115 121 109 117 104 107 102 100 .+-. 12*
control) in T.sub.2 seed p-value** 0.04 0.01 0.11 0.02 0.17 0.14
0.19 N/A Oil content (% 100 .+-. 1 102 .+-. 2 101 .+-. 4 105 .+-. 1
101 .+-. 2 No No 100 .+-. 4 control) in T.sub.3 data data seed
p-value** 0.72 0.18 0.79 0.01 0.60 No No N/A data data *Population
mean of the oil content in seed from transgenic lines planted
within 30 days of ME00072. Variation is presented as the standard
error of the mean. **The p-values for T.sub.2 seed were calculated
using z-scores. ***The p-values for T.sub.3 seed were calculated
using a Student's t-test.
[0371] The oil content in T.sub.3 seed from one event of ME00072
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 11, the oil
content was increased to 105% in seed from event -06 compared to
the oil content in control seed.
Example 10
Results for ME00085 Events
[0372] T.sub.2 and T.sub.3 seed from six events and four events,
respectively, of ME00085 containing Ceres Clone 4829 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0373] The oil content in T.sub.2 seed from two events of ME00085
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME00085. As presented in Table 12, the oil content was increased to
120% and 117% in seed from events -01 and -02, respectively,
compared to the population mean.
TABLE-US-00012 TABLE 12 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME00085 events containing Ceres Clone 4829 Event
Event Event Event Event Event -01 -02 -03 -04 -05 -06 Control Oil
content (% 120 117 92 98 100 109 100 .+-. 12* control) in T.sub.2
seed p-value** 0.01 0.03 0.13 0.19 0.19 0.11 N/A Oil content (% 95
.+-. 4 111 .+-. 2 111 .+-. 2 104 .+-. 4 No data No data 100 .+-. 4
control) in T.sub.3 seed p-value*** 0.20 <0.01 <0.01 0.09 No
data No data N/A *Population mean of the oil content in seed from
transgenic lines planted within 30 days of ME00085. Variation is
presented as the standard error of the mean. **The p-values for
T.sub.2 seed were calculated using z-scores. ***The p-values for
T.sub.3 seed were calculated using a Student's t-test.
[0374] The oil content in T.sub.3 seed from two events of ME00085
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 12, the oil
content was increased to 111% in seed from events -02 and -03
compared to the oil content in control seed.
Example 11
Results for ME00147 Events
[0375] T.sub.2 and T.sub.3 seed from seven events and five events,
respectively, of ME00147 containing Ceres Clone 28635 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0376] The oil content in T.sub.2 seed from three events of ME00147
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME00147. As presented in Table 13, the oil content was increased to
119%, 120%, and 118% in seed from events -02, -03, and -04,
respectively, compared to the population mean.
TABLE-US-00013 TABLE 13 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME00147 events containing Ceres Clone 28635 Event
Event Event Event Event Event Event Event -02 -03 -04 -05 -06 -07
-08 -09 Control Oil content 119 120 118 No 95 108 105 102 100 .+-.
12* (% control) data in T.sub.2 seed p-value** 0.02 0.01 0.02 No
0.16 0.13 0.16 0.19 N/A data Oil content 102 .+-. 2 101 .+-. 0 105
.+-. 2 99 .+-. 2 97 .+-. 4 No No No 100 .+-. 4 (% control) data
data data in T.sub.3 seed p-value*** 0.15 0.47 0.04 0.52 0.21 No No
No N/A data data data *Population mean of the oil content in seed
from transgenic lines planted within 30 days of ME00147. Variation
is presented as the standard error of the mean. **The p-values for
T.sub.2 seed were calculated using z-scores. ***The p-values for
T.sub.3 seed were calculated using a Student's t-test.
[0377] The oil content in T.sub.3 seed from one event of ME00147
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 13, the oil
content was increased to 105% in seed from event -04 compared to
the oil content in control seed.
Example 12
Results for ME00896 Events
[0378] T.sub.2 and T.sub.3 seed from three events and four events,
respectively, of ME00896 containing Ceres Clone 16403 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0379] The oil content in T.sub.2 seed from three events of ME00896
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME00896. As presented in Table 14, the oil content was increased to
130%, 126%, and 118% in seed from events -03, -04, and -05,
respectively, compared to the population mean.
TABLE-US-00014 TABLE 14 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME00896 events containing Ceres Clone 16403 Event
-02 Event -03 Event -04 Event -05 Control Oil No data 130 126 118
100 .+-. 10* content (% control) in T.sub.2 seed p-value** No data
0.01 0.02 0.05 N/A Oil 102 .+-. 2 100 .+-. 1 104 .+-. 2 100 .+-. 1
100 .+-. 4 content (% control) in T.sub.3 seed p-value*** 0.09 0.91
0.03 0.93 N/A *Population mean of the oil content in seed from
transgenic lines planted within 30 days of ME00896. Variation is
presented as the standard error of the mean. **The p-values for
T.sub.2 seed were calculated using z-scores. ***The p-values for
T.sub.3 seed were calculated using a Student's t-test.
[0380] The oil content in T.sub.3 seed from one event of ME00896
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 14, the oil
content was increased to 104% in seed from event -04 compared to
the oil content in control seed.
Example 13
Results for ME00900 Events
[0381] T.sub.2 and T.sub.3 seed from three events and two events,
respectively, of ME00900 containing Ceres Clone 41046 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0382] The oil content in T.sub.2 seed from two events of ME00900
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME00900. As presented in Table 15, the oil content was increased to
132% and 128% in seed from events -01 and -02, respectively,
compared to the population mean.
TABLE-US-00015 TABLE 15 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME00900 events containing Ceres Clone 41046 Event
-01 Event -02 Event -03 Control Oil 132 128 108 100 .+-. 10*
content (% control) in T.sub.2 seed p-value** 0.01 0.01 0.11 N/A
Oil 105 .+-. 3 No data 97 .+-. 2 100 .+-. 4 content (% control) in
T.sub.3 seed p-value*** 0.01 No data 0.06 N/A *Population mean of
the oil content in seed from transgenic lines planted within 30
days of ME00900. Variation is presented as the standard error of
the mean. **The p-values for T.sub.2 seed were calculated using
z-scores. ***The p-values for T.sub.3 seed were calculated using a
Student's t-test.
[0383] The oil content in T.sub.3 seed from one event of ME00900
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 15, the oil
content was increased to 105% in seed from event -01 compared to
the oil content in control seed.
Example 14
Results for ME00913 Events
[0384] T.sub.2 and T.sub.3 seed from four events of ME00913
containing Ceres Clone 19244 was analyzed for oil content using
FT-NIR spectroscopy as described in Example 2.
[0385] The oil content in T.sub.2 seed from two events of ME00913
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME00913. As presented in Table 16, the oil content was increased to
130% and 128% in seed from events -01 and -04, respectively,
compared to the population mean.
TABLE-US-00016 TABLE 16 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME00913 events containing Ceres Clone 19244 Event
-01 Event -02 Event -03 Event -04 Control Oil 130 114 91 128 100
.+-. 10* content (% control) in T.sub.2 seed p-value** 0.01 0.08
0.11 0.01 N/A Oil 99 .+-. 5 107 .+-. 0 104 .+-. 2 106 .+-. 4 100
.+-. 4 content (% control) in T.sub.3 seed p-value*** 0.91 <0.01
0.01 0.02 N/A *Population mean of the oil content in seed from
transgenic lines planted within 30 days of ME00913. Variation is
presented as the standard error of the mean. **The p-values for
T.sub.2 seed were calculated using z-scores. ***The p-values for
T.sub.3 seed were calculated using a Student's t-test.
[0386] The oil content in T.sub.3 seed from three events of ME00913
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 16, the oil
content was increased to 107%, 104%, and 106% in seed from events
-02, -03, and -04, respectively, compared to the oil content in
control seed.
Example 15
Results for ME01704 Events
[0387] T.sub.2 and T.sub.3 seed from four events and five events,
respectively, of ME01704 containing Ceres Clone 7894 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0388] The oil content in T.sub.2 seed from three events of ME01704
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME01704. As presented in Table 17, the oil content was increased to
126%, 118%, and 132% in seed from events -02, -04, and -06,
respectively, compared to the population mean.
TABLE-US-00017 TABLE 17 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME01704 events containing Ceres Clone 7894 Event
Event Event Event Event -01 -02 -03 -04 -06 Control Oil content 103
126 No data 118 132 100 .+-. 9* (% control) in T.sub.2 seed
p-value** 0.17 0.01 No data 0.04 <0.01 N/A Oil content 101 .+-.
1 103 .+-. 2 97 .+-. 6 109 .+-. 3 107 .+-. 3 100 .+-. 4 (% control)
in T.sub.3 seed p-value*** 0.60 0.12 0.49 <0.01 <0.01 N/A
*Population mean of the oil content in seed from transgenic lines
planted within 30 days of ME01704. Variation is presented as the
standard error of the mean. **The p-values for T.sub.2 seed were
calculated using z-scores. ***The p-values for T.sub.3 seed were
calculated using a Student's t-test.
[0389] The oil content in T.sub.3 seed from two events of ME01704
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 17, the oil
content was increased to 109% and 107% in seed from events -04 and
-06, respectively, compared to the oil content in control seed.
Example 16
Results for ME02505 Events
[0390] T.sub.2 and T.sub.3 seed from five events of ME02505
containing Ceres Clone 158765 was analyzed for oil content using
FT-NIR spectroscopy as described in Example 2.
[0391] The oil content in T.sub.2 seed from three events of ME02505
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME02505. As presented in Table 18, the oil content was increased to
120%, 129%, and 140% in seed from events -01, -02, and -03,
respectively, compared to the population mean.
TABLE-US-00018 TABLE 18 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME02505 events containing Ceres Clone 158765
Event Event Event Event Event -01 -02 -03 -04 -05 Control Oil
content 120 129 140 104 105 100 .+-. 11* (% control) in T.sub.2
seed p-value** 0.02 <0.01 <0.01 0.19 0.19 N/A Oil content 101
.+-. 1 100 .+-. 1 103 .+-. 1 108 .+-. 1 105 .+-. 2 100 + 4 (%
control) in T.sub.3 seed p-value*** 0.47 0.79 0.01 <0.01 0.02
N/A *Population mean of the oil content in seed from transgenic
lines planted within 30 days of ME02505. Variation is presented as
the standard error of the mean. **The p-values for T.sub.2 seed
were calculated using z-scores. ***The p-values for T.sub.3 seed
were calculated using a Student's t-test.
[0392] The oil content in T.sub.3 seed from three events of ME02505
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 18, the oil
content was increased to 103%, 108%, and 105% in seed from events
-03, -04, and -05, respectively, compared to the oil content in
control seed.
Example 17
Results for ME02525 Events
[0393] T.sub.2 and T.sub.3 seed from four events of ME02525
containing Ceres Clone 121021 was analyzed for oil content using
FT-NIR spectroscopy as described in Example 2.
[0394] The oil content in T.sub.2 seed from two events of ME02525
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME02525. As presented in Table 19, the oil content was increased to
134% and 145% in seed from events -01 and -02, respectively,
compared to the population mean.
TABLE-US-00019 TABLE 19 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME02525 events containing Ceres Clone 121021
Event Event Event Event Event -01 -02 -03 -04 -07 Control Oil
content 134 145 113 No data 103 100 .+-. 11* (% control) in T.sub.2
seed p-value** <0.01 <0.01 0.09 No data 0.20 N/A Oil content
102 .+-. 2 99 .+-. 1 98 .+-. 1 103 .+-. 2 No data 100 .+-. 4 (%
control) in T.sub.3 seed p-value*** 0.19 0.16 0.10 0.05 No data N/A
*Population mean of the oil content in seed from transgenic lines
planted within 30 days of ME02525. Variation is presented as the
standard error of the mean. **The p-values for T.sub.2 seed were
calculated using z-scores. ***The p-values for T.sub.3 seed were
calculated using a Student's t-test.
[0395] The oil content in T.sub.3 seed from one event of ME02525
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 19, the oil
content was increased to 103% in seed from event -04 compared to
the oil content in control seed.
Example 18
Results for ME00902 Events
[0396] T.sub.2 and T.sub.3 seed from four events of ME00902
containing Ceres Clone
[0397] 36412 was analyzed for oil content using FT-NIR spectroscopy
as described in Example 2.
[0398] The oil content in T.sub.2 seed from ME00902 events was not
observed to differ significantly from the mean oil content in seed
from transgenic Arabidopsis lines planted within 30 days of ME00902
(Table 20).
TABLE-US-00020 TABLE 20 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME00902 events containing Ceres Clone 36412 Event
-01 Event -03 Event -04 Event -05 Control Oil 115 114 114 113 100
.+-. 10* content (% control) in T.sub.2 seed p-value** 0.07 0.07
0.08 0.08 N/A Oil 104 .+-. 1 98 .+-. 1 102 .+-. 1 103 .+-. 3 100
.+-. 4 content (% control) in T.sub.3 seed p-value*** <0.01 0.04
0.09 0.12 N/A *Population mean of the oil content in seed from
transgenic lines planted within 30 days of ME00902. Variation is
presented as the standard error of the mean. **The p-values for
T.sub.2 seed were calculated using z-scores. ***The p-values for
T.sub.3 seed were calculated using a Student's t-test.
[0399] The oil content in T.sub.3 seed from one event of ME00902
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 20, the oil
content was increased to 104% in seed from event -01 compared to
the oil content in control seed. The oil content in T.sub.3 seed
from one event of ME00902 was significantly decreased compared to
the oil content in corresponding control seed. As presented in
Table 20, the oil content was decreased to 98% in seed from event
-03 compared to the oil content in control seed.
Example 19
Results for ME00914 Events
[0400] T.sub.2 and T.sub.3 seed from five events and four events,
respectively, of ME00914 containing Ceres Clone 8161 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0401] The oil content in T.sub.2 seed from four events of ME00914
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME00914. As presented in Table 21, the oil content was increased to
121%, 122%, 125%, and 129% in seed from events -01, -02, -03, and
-05, respectively, compared to the population mean.
TABLE-US-00021 TABLE 21 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME00914 events containing Ceres Clone 8161 Event
Event Event Event Event -01 -02 -03 -04 -05 Control Oil content 121
122 125 116 129 100 .+-. 10* (% control) in T.sub.2 seed p-value**
0.04 0.03 0.02 0.06 0.01 N/A Oil content 101 .+-. 0 98 .+-. 1 98
.+-. 2 No data 107 .+-. 1 100 .+-. 4 (% control) in T.sub.3 seed
p-value*** 0.46 <0.01 0.05 No data <0.01 N/A *Population mean
of the oil content in seed from transgenic lines planted within 30
days of ME00914. Variation is presented as the standard error of
the mean. **The p-values for T.sub.2 seed were calculated using
z-scores. ***The p-values for T.sub.3 seed were calculated using a
Student's t-test.
[0402] The oil content in T.sub.3 seed from one event of ME00914
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 21, the oil
content was increased to 107% in seed from event -05 compared to
the oil content in control seed. The oil content in T.sub.3 seed
from two events of ME00914 was significantly decreased compared to
the oil content in corresponding control seed. As presented in
Table 21, the oil content was decreased to 98% in seed from events
-02 and -03 compared to the oil content in control seed.
Example 20
Results for ME01754 Events
[0403] T.sub.2 and T.sub.3 seed from five events and four events,
respectively, of ME01754 containing Ceres Clone 368 was analyzed
for oil content using FT-NIR spectroscopy as described in Example
2.
[0404] The oil content in T.sub.2 seed from two events of ME01754
was significantly increased compared to the mean oil content in
seed from transgenic Arabidopsis lines planted within 30 days of
ME01754. As presented in Table 22, the oil content was increased to
124% and 131% in seed from events -02 and -04, respectively,
compared to the population mean.
TABLE-US-00022 TABLE 22 Oil content (% control) in T.sub.2 and
T.sub.3 seed from ME01754 events containing Ceres Clone 368 Event
Event Event Event Event -01 -02 -03 -04 -05 Control Oil content 100
124 113 131 89 100 .+-. 9* (% control) in T.sub.2 seed p-value**
0.18 0.01 0.08 <0.01 0.10 N/A Oil content 97 .+-. 2 103 .+-. 2
109 .+-. 3 96 .+-. 1 No data 100 .+-. 4 (% control) in T.sub.3 seed
p-value*** 0.11 0.02 0.10 <0.01 No data N/A *Population mean of
the oil content in seed from transgenic lines planted within 30
days of ME01754. Variation is presented as the standard error of
the mean. **The p-values for T.sub.2 seed were calculated using
z-scores. ***The p-values for T.sub.3 seed were calculated using a
Student's t-test.
[0405] The oil content in T.sub.3 seed from one event of ME01754
was significantly increased compared to the oil content in
corresponding control seed. As presented in Table 22, the oil
content was increased to 103% in seed from event -02 compared to
the oil content in control seed. The oil content in T.sub.3 seed
from one event of ME01754 was significantly decreased compared to
the oil content in corresponding control seed. As presented in
Table 22, the oil content was decreased to 96% in seed from event
-04 compared to the oil content in control seed.
Example 21
Determination of Functional Homolog and/or Ortholog Sequences
[0406] A subject sequence was considered a functional homolog or
ortholog of a query sequence if the subject and query sequences
encoded proteins having a similar function and/or activity. A
process known as Reciprocal BLAST (Rivera et al., Proc. Natl. Acad.
Sci. USA, 95:6239-6244 (1998)) was used to identify potential
functional homolog and/or ortholog sequences from databases
consisting of all available public and proprietary peptide
sequences, including NR from NCBI and peptide translations from
Ceres clones.
[0407] Before starting a Reciprocal BLAST process, a specific query
polypeptide was searched against all peptides from its source
species using BLAST in order to identify polypeptides having BLAST
sequence identity of 80% or greater to the query polypeptide and an
alignment length of 85% or greater along the shorter sequence in
the alignment. The query polypeptide and any of the aforementioned
identified polypeptides were designated as a cluster.
[0408] The BLASTP version 2.0 program from Washington University at
Saint Louis, Mo., USA was used to determine BLAST sequence identity
and E-value. The BLASTP version 2.0 program includes the following
parameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5;
and 3) the -postsw option. The BLAST sequence identity was
calculated based on the alignment of the first BLAST HSP
(High-scoring Segment Pairs) of the identified potential functional
homolog and/or ortholog sequence with a specific query polypeptide.
The number of identically matched residues in the BLAST HSP
alignment was divided by the HSP length, and then multiplied by 100
to get the BLAST sequence identity. The HSP length typically
included gaps in the alignment, but in some cases gaps were
excluded.
[0409] The main Reciprocal BLAST process consists of two rounds of
BLAST searches; forward search and reverse search. In the forward
search step, a query polypeptide sequence, "polypeptide A," from
source species SA was BLASTed against all protein sequences from a
species of interest. Top hits were determined using an E-value
cutoff of 10.sup.-5 and a sequence identity cutoff of 35%. Among
the top hits, the sequence having the lowest E-value was designated
as the best hit, and considered a potential functional homolog or
ortholog. Any other top hit that had a sequence identity of 80% or
greater to the best hit or to the original query polypeptide was
considered a potential functional homolog or ortholog as well. This
process was repeated for all species of interest.
[0410] In the reverse search round, the top hits identified in the
forward search from all species were BLASTed against all protein
sequences from the source species SA. A top hit from the forward
search that returned a polypeptide from the aforementioned cluster
as its best hit was also considered as a potential functional
homolog or ortholog.
[0411] Functional homologs and/or orthologs were identified by
manual inspection of potential functional homolog and/or ortholog
sequences. Representative functional homologs and/or orthologs for
SEQ ID NO:81, SEQ ID NO:94, SEQ ID NO:111, SEQ ID NO:136, SEQ ID
NO:152, SEQ ID NO:159, SEQ ID NO:171, SEQ ID NO:176, SEQ ID NO:178,
SEQ ID NO:193, SEQ ID NO:332, SEQ ID NO:342, SEQ ID NO:344, SEQ ID
NO:346, SEQ ID NO:359, SEQ ID NO:374, and SEQ ID NO:398 are shown
in FIGS. 1-13, respectively. The percent identities of functional
homologs and/or orthologs to SEQ ID NO:81, SEQ ID NO:94, SEQ ID
NO:111, SEQ ID NO:136, SEQ ID NO:152, SEQ ID NO:159, SEQ ID NO:171,
SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:193, SEQ ID NO:332, SEQ ID
NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:359, SEQ ID NO:374,
and SEQ ID NO:398 are shown below in Tables 23-34, respectively.
The BLAST sequence identities and E-values given in Tables 23-34
were taken from the forward search round of the Reciprocal BLAST
process.
TABLE-US-00023 TABLE 23 Percent identity to Ceres Clone 41573 (SEQ
ID NO: 81) SEQ ID % Iden- Designation Species NO: tity e-value
Ceres CLONE Zea mays 82 54.9 2.49E-82 ID no. 1560908 Public GI no.
Nicotiana tabacum 83 54.6 8.99E-85 2739168 Ceres CLONE Zea mays 84
54.5 1.19E-82 ID no. 399052 Ceres CLONE Triticum aestivum 85 54.2
1.49E-82 ID no. 1314177 Public GI no. Nicotiana tabacum 86 53.9
2.09E-85 15824567 Ceres CLONE Glycine max 87 53.2 2.90E-79 ID no.
1371577 Public GI no. Nicotiana tabacum 88 53.2 1.90E-84 15824565
Public GI no. Oryza sativa subsp. 89 53.2 1.80E-77 50920801
japonica Public GI no. Oryza sativa subsp. 90 51.8 7.20E-76
50909807 japonica Ceres CLONE Triticum aestivum 91 51.2 1.20E-75 ID
no. 639223 Public GI no. Oryza sativa 92 51.2 1.80E-72 37531218
Ceres CLONE Populus balsamifera 502 63.8 2.00E-96 ID no. 1476735
subsp. trichocarpa
TABLE-US-00024 TABLE 24 Percent identity to Ceres Clone 25429 (SEQ
ID NO: 94) SEQ ID % Iden- Designation Species NO: tity e-value
Ceres Annot: Populus balsamifera 96 75 1.70E-35 1488311_PRT subsp.
trichocarpa Ceres CLONE Brassica napus 97 74.7 1.49E-34 ID no.
953928 Ceres CLONE Glycine max 98 74.5 3.60E-33 ID no. 524682 Ceres
CLONE Parthenium argentatum 99 73.4 1.29E-28 ID no. 1609735 Ceres
CLONE Brassica napus 100 73.4 2.40E-36 ID no. 949174 Ceres CLONE
Brassica napus 101 72.5 3.90E-36 ID no. 1299820 Public GI no.
Hyacinthus orientalis 102 72.5 1.30E-35 42565379 Ceres CLONE Zea
mays 103 71.6 2.80E-35 ID no. 426736 Ceres CLONE Brassica napus 104
71.4 1.39E-33 ID no. 1094375 Ceres CLONE Glycine max 105 71.1
2.89E-33 ID no. 691062 Public GI no. Hyacinthus orientalis 106 70.7
2.49E-34 47026878 Public GI no. Prunus dulcis 107 69.9 8.20E-36
24473796 Ceres Annot: Populus balsamifera 109 67.2 3.39E-30
1465437_PRT subsp. trichocarpa Ceres CLONE Gossypium hirsutum 503
76.7 7.99E-38 ID no. 1798334 Ceres CLONE Gossypium hirsutum 504
76.3 9.50E-35 ID no. 1886478 Ceres CLONE Musa acuminate 505 72.9
6.89E-32 ID no. 1727128 Public GI no. Parthenium argentatum 506 66
3.30E-32 730583
TABLE-US-00025 TABLE 25 Percent identity to Ceres Clone 5750 (SEQ
ID NO: 111) SEQ ID % Iden- Designation Species NO: tity e-value
Public GI no. Glycine max 112 96.5 1.20E-41 42362268 Ceres CLONE
Glycine max 113 96.5 1.20E-41 ID no. 709027 Ceres CLONE Glycine max
114 96.5 1.20E-41 ID no. 853298 Ceres CLONE Glycine max 115 96.5
1.20E-41 ID no. 1417425 Public GI no. Populus tremula .times. 116
94.8 9.09E-37 27435806 Populus tremuloides Ceres Annot: Populus
balsamifera 118 94.8 9.09E-37 1481954_PRT subsp. trichocarpa Public
GI no. Ipomoea trifida 119 94.2 5.19E-41 45935118 Ceres CLONE
Triticum aestivum 120 93.8 3.79E-38 ID no. 1017141 Ceres CLONE Zea
mays 121 93.8 3.79E-38 ID no. 1448636 Public GI no. Oryza sativa
subsp. 122 93.8 3.79E-38 50919707 japonica Public GI no. Triticum
aestivum 123 93.8 3.79E-38 40641585 Public GI no. Arabidopsis
thaliana 124 93.75 4.39E-37 38566522 Ceres CLONE Arabidopsis
thaliana 125 93.75 4.39E-37 ID no. 1338131 Ceres CLONE Zea mays 126
90.8 9.79E-40 ID no. 300692 Ceres CLONE Brassica napus 127 90.8
3.30E-39 ID no. 947192 Ceres CLONE Zea mays 128 90.8 8.80E-39 ID
no. 1465004 Ceres CLONE Brassica napus 129 89.6 6.19E-38 ID no.
1122958 Ceres CLONE Brassica napus 130 86.4 9.69E-33 ID no. 944737
Ceres CLONE Zea mays 131 85.1 5.80E-35 ID no. 217797 Ceres CLONE
Glycine max 132 85.1 5.80E-35 ID no. 520185 Public GI no.
Ostreococcus tauri 133 83 2.09E-30 55978016 Ceres CLONE Zea mays
134 82.7 2.49E-34 ID no. 1436585 Ceres CLONE Panicum virgatum 507
96.1 1.70E-37 ID no. 1777369 Ceres CLONE Musa acuminata 508 95
5.49E-39 ID no. 1744578 Ceres CLONE Gossypium hirsutum 509 94.2
1.80E-40 ID no. 100008703 Ceres CLONE Musa acuminata 510 93.8
1.10E-38 ID no. 1723582
TABLE-US-00026 TABLE 26 Percent identity to Ceres Clone 218626 (SEQ
ID NO: 136) SEQ ID % Iden- Designation Species NO: tity e-value
Public GI no. Oryza sativa subsp. 137 91.9 3.90E-219 30409136
indica Public GI no. Oryza sativa subsp. 138 91 3.29E-222 50940751
japonica Ceres CLONE Zea mays 139 84.9 3.70E-207 ID no. 1571117
Ceres CLONE Zea mays 140 83.9 2.50E-201 ID no. 424395 Ceres Annot:
Populus balsamifera 142 81.8 1.70E-161 1440705_PRT subsp.
trichocarpa Ceres Annot: Populus balsamifera 144 81.6 4.19E-174
1493584_PRT subsp. trichocarpa Ceres Annot: Populus balsamifera 146
79.2 1.80E-159 1463076_PRT subsp. trichocarpa Ceres CLONE
Arabidopsis thaliana 147 79.1 9.99E-171 ID no. 1002421 Ceres Annot:
Populus balsamifera 149 78.6 1.20E-153 1516369_PRT subsp.
trichocarpa Public GI no. Arabidopsis thaliana 150 78.3 3.40E-188
30693666 Ceres CLONE Panicum virgatum 511 85.8 1.19E-208 ID no.
1796001
TABLE-US-00027 TABLE 27 Percent identity to Ceres Clone 121021 (SEQ
ID NO: 152) SEQ ID % Iden- Designation Species NO: tity e-value
Ceres Annot: Populus balsamifera 154 57.6 1.79E-17 1501628_PRT
subsp. trichocarpa Ceres Annot: Populus balsamifera 156 52.8
7.69E-24 1519046_PRT subsp. trichocarpa Ceres CLONE Glycine max 157
51.7 9.09E-21 ID no. 1121512
TABLE-US-00028 TABLE 28 Percent identity to Ceres Clone 158765 (SEQ
ID NO: 159) SEQ ID % Iden- Designation Species NO: tity e-value
Public GI no. Arabidopsis thaliana 160 97.9 5.19E-73 32562996
Public GI no. Lycopersicon 161 66.1 5.00E-36 5669656 esculentum
Ceres CLONE Triticum aestivum 162 65.3 6.99E-30 ID no. 754061 Ceres
CLONE Triticum aestivum 163 65.3 6.99E-30 ID no. 1329861 Ceres
CLONE Glycine max 164 65.1 1.10E-40 ID no. 537752 Ceres CLONE
Triticum aestivum 165 64.2 1.90E-29 ID no. 1322549 Ceres CLONE Zea
mays 166 62.2 4.90E-29 ID no. 282892 Ceres CLONE Zea mays 167 62.2
1.29E-28 ID no. 284046 Ceres CLONE Zea mays 168 61.6 4.90E-29 ID
no. 1388825 Public GI no. Oryza sativa subsp. 169 50 1.60E-30
50925813 japonica Ceres CLONE Gossypium hirsutum 545 72.8 5.09E-36
ID no. 1839717
TABLE-US-00029 TABLE 29 Percent identity to Ceres Clone 16403 (SEQ
ID NO: 171) SEQ ID % Iden- Designation Species NO: tity e-value
Ceres CLONE Glycine max 172 58.9 7.29E-67 ID no. 611156 Ceres
Annot: Populus balsamifera 174 56.2 4.39E-60 1464944_PRT subsp.
trichocarpa Ceres CLONE Musa acuminata 530 60.2 1.70E-58 ID no.
1728680 Ceres CLONE Gossypium hirsutum 531 59.1 8.00E-70 ID no.
1807796 Ceres CLONE Panicum virgatum 532 52.3 4.69E-56 ID no.
1771837 Ceres CLONE Panicum virgatum 533 51.4 4.69E-56 ID no.
1773482
TABLE-US-00030 TABLE 30 Percent identity to Ceres Clone 28635 (SEQ
ID NO: 178) SEQ ID % Iden- Designation Species NO: tity e-value
Public GI no. Glycine max 179 80.6 1.80E-180 2463569 Ceres Annot:
Populus balsamifera 181 79.9 6.20E-157 1514021_PRT subsp.
trichocarpa Public GI no. Centella asiatica 182 79.8 2.49E-176
55710094 Ceres Annot: Populus balsamifera 184 79.7 2.90E-166
1503464_PRT subsp. trichocarpa Public GI no. Panax notoginseng 185
78.8 2.19E-177 75859951 Public GI no. Glycyrrhiza glabra 186 78.8
5.19E-176 1449163 Public GI no. Glycyrrhiza glabra 187 78.1
3.69E-175 1449165 Public GI no. Lotus japonicus 188 78.1 1.60E-172
28208268 Public GI no. Panax ginseng 189 77.8 3.69E-175 41224629
Public GI no. Medicago truncatula 190 77.8 2.10E-172 27475614
Public GI no. Artemisia annua 191 77 8.10E-171 38426486 Ceres CLONE
Gossypium hirsutum 534 79.2 9.80E-175 ID no. 1920025 Public GI no.
Polygala tenuifolia 535 78.6 2.29E-175 110293133 Public GI no.
Solanum tuberosum 536 76.0 1.5E-171 5360655 Public GI no. Capsicum
annuum 537 75.7 2.2E-170 4426953 Public GI no. Nicotiana tabacum
538 75.5 1.9E-171 1552717 Public GI no. Bupleurum falcatum 539 75.9
1.69E-170 66393825 Public GI no. Nicotiana benthamiana 540 74.8
2.5E-169 1706774 Ceres CLONE Panicum virgatum 541 69.8 3.20E-153 ID
no. 1749989 Public GI no. Oryza sativa subsp. 542 68.6 1.60E-149
115456049 japonica Public GI no. Zea mays 543 68.3 3.79E-150
2463567 Ceres CLONE Triticum aestivum 544 65.1 5.9E-145 ID no.
706088
TABLE-US-00031 TABLE 31 Percent identity to Ceres Clone 35698 (SEQ
ID NO: 193) SEQ ID % Iden- Designation Species NO: tity e-value
Ceres CLONE Arabidopsis thaliana 194 98.6 1.90E-36 ID no. 1346445
Public GI no. Lycopersicon esculentum 195 98 8.20E-52 441457 Public
GI no. Arabidopsis thaliana 196 98 3.49E-51 19347859 Ceres Annot:
Populus balsamifera 198 97 2.80E-51 1483290_PRT subsp. trichocarpa
Public GI no. Capsicum annuum 199 97 1.29E-51 40287554 Public GI
no. Arabidopsis thaliana 200 97 2.20E-51 21553796 Public GI no.
Gossypium thurberi 201 97 2.20E-51 28569271 Public GI no. Gossypium
hirsutum 202 97 2.20E-51 28569265 Public GI no. Arabidopsis
thaliana 203 97 2.20E-51 66354420 Public GI no. Arabidopsis
thaliana 204 97 2.20E-51 21280893 Public GI no. Arabidopsis
thaliana 205 97 2.20E-51 21554343 Public GI no. Glycine max 206 97
4.50E-51 22597164 Ceres CLONE Triticum aestivum 207 96 9.40E-51 ID
no. 617835 Public GI no. Arabidopsis thaliana 208 96 8.40E-50
30693871 Public GI no. Mesembryanthemum 209 96 4.50E-51 5762457
crystallinum Public GI no. Lycopersicon esculentum 210 96 4.50E-51
464981 Public GI no. Pisum sativum 211 96 4.50E-51 456568 Public GI
no. Solanum tuberosum 212 96 3.49E-51 77416935 Public GI no.
Gossypium arboreum 213 96 3.49E-51 28569267 Public GI no. Gossypium
hirsutum 214 96 3.49E-51 28569261 Ceres CLONE Arabidopsis thaliana
215 95.8 9.99E-36 ID no. 39130 Ceres CLONE Arabidopsis thaliana 216
95.8 9.99E-36 ID no. 16865 Ceres CLONE Glycine max 217 95.8
1.30E-35 ID no. 575067 Ceres Annot: Populus balsamifera 219 95.8
1.30E-35 1467392_PRT subsp. trichocarpa Ceres CLONE Arabidopsis
thaliana 220 95.8 9.99E-36 ID no. 25162 Ceres Annot: Populus
balsamifera 222 95.8 8.20E-36 1529647_PRT subsp. trichocarpa Ceres
CLONE Zea mays 223 95.8 8.20E-36 ID no. 1405728 Public GI no. Picea
abies 224 95.3 9.99E-31 54288726 Public GI no. Oryza sativa subsp.
225 95 1.20E-50 50906823 japonica Public GI no. Oryza sativa subsp.
226 95 3.19E-50 83306206 indica Public GI no. Oryza sativa subsp.
227 95 1.89E-50 20152203 japonica Public GI no. Capsicum annuum 228
94.5 2.20E-35 40287568 Public GI no. Oryza sativa subsp. 229 94.1
4.00E-50 50929483 japonica Ceres Annot: Populus balsamifera 231
93.1 4.50E-35 1450556_PRT subsp. trichocarpa Ceres CLONE Triticum
aestivum 232 93.1 8.40E-50 ID no. 1031152 Public GI no. Triticum
aestivum 233 93.1 8.40E-50 52548244 Public GI no. Solanum tuberosum
234 92.8 7.60E-33 82621144 Ceres CLONE Triticum aestivum 235 92.1
2.80E-49 ID no. 878043 Public GI no. Oryza sativa subsp. 236 92.1
2.80E-49 34909292 japonica Public GI no. Zea mays 237 92.1 2.80E-49
2668744 Ceres CLONE Glycine max 238 91.7 9.40E-35 ID no. 511132
Ceres Annot: Populus balsamifera 240 91.7 1.19E-34 1533930_PRT
subsp. trichocarpa Ceres Annot: Populus balsamifera 242 91.1
9.99E-38 1495171_PRT subsp. trichocarpa Public GI no. Oryza sativa
243 90.5 3.19E-50 20086317 Ceres CLONE Arabidopsis thaliana 244
90.4 4.09E-34 ID no. 10022 Ceres CLONE Arabidopsis thaliana 245
90.4 4.09E-34 ID no. 12547 Ceres CLONE Arabidopsis thaliana 246
90.4 4.09E-34 ID no. 27679 Public GI no. Arabidopsis thaliana 247
90.1 9.60E-49 20259611 Public GI no. Hordeum vulgare 248 90.1
5.30E-48 30025160 Public GI no. Oryza sativa subsp. 249 89.2
9.60E-49 50904839 japonica Ceres CLONE Zea mays 250 89.2 8.69E-48
ID no. 1063753 Ceres Annot: Populus balsamifera 252 89 1.09E-33
1533218_PRT subsp. trichocarpa Public GI no. Arabidopsis thaliana
253 88.8 2.89E-33 297880 Ceres CLONE Zea mays 254 88.2 1.09E-47 ID
no. 1357060 Public GI no. Arachis hypogaea 255 87.2 2.89E-47
54402104 Public GI no. Oryza sativa subsp. 256 87.2 1.29E-46
50725323 japonica Ceres CLONE Zea mays 257 87.2 9.89E-47 ID no.
376667 Ceres CLONE Zea mays 258 86.8 1.50E-41 ID no. 256705 Public
GI no. Arabidopsis thaliana 259 86.2 4.80E-47 20259629 Public GI
no. Plantago major 260 86.2 6.09E-47 52851174 Ceres CLONE Zea mays
261 86.2 1.60E-46 ID no. 1061097 Public GI no. Oryza sativa 262
86.2 4.39E-44 1373001 Public GI no. Arabidopsis thaliana 263 86.2
9.89E-47 66354468 Public GI no. Pinus resinosa 264 82.3 2.40E-45
4100646 Ceres CLONE Zea mays 265 98 2.80E-51 ID no. 1380019 Ceres
CLONE Artificial Sequence 266 98.04 4.80E-56 ID no. 1380019_T Ceres
CLONE Artificial Sequence 267 98.63 3.30E-41 ID no. 1346445_T
Public GI no. Artificial Sequence 268 98.04 1.40E-56 441457_T
Public GI no. Artificial Sequence 269 98.04 6.10E-56 19347859_T
Ceres Annot: Populus balsamifera 270 97.06 4.80E-56 1483290_T
subsp. trichocarpa Public GI no. Artificial Sequence 271 97.06
2.30E-56 40287554_T Public GI no. Artificial Sequence 272 97.06
3.70E-56 21553796_T Public GI no. Artificial Sequence 273 97.06
3.70E-56 28569271_T Public GI no. Artificial Sequence 274 97.06
3.70E-56 28569265_T Public GI no. Artificial Sequence 275 97.06
3.70E-56 66354420_T Public GI no. Artificial Sequence 276 97.06
3.70E-56 21280893_T Public GI no. Artificial Sequence 277 97.06
3.70E-56 21554343_T Public GI no. Artificial Sequence 278 97.03
7.80E-56 22597164_T Ceres CLONE Artificial Sequence 279 96.08
1.60E-55 ID no. 617835_T Public GI no. Artificial Sequence 280
96.08 1.50E-54 30693871_T Public GI no. Artificial Sequence 281
96.08 7.80E-56 5762457_T Public GI no. Artificial Sequence 282
96.08 7.80E-56 464981_T Public GI no. Artificial Sequence 283 96.08
7.80E-56 456568_T Public GI no. Artificial Sequence 284 96.08
6.10E-56 77416935_T Public GI no. Artificial Sequence 285 96.08
6.10E-56 28569267_T Public GI no. Artificial Sequence 286 96.08
6.10E-56 28569261_T Ceres CLONE Artificial Sequence 287 95.89
1.80E-40 ID no. 39130_T Ceres CLONE Artificial Sequence 288 95.89
1.80E-40 ID no. 16865_T Ceres CLONE Artificial Sequence 289 95.89
2.30E-40 ID no. 575067_T Ceres Annot: Populus balsamifera 290 95.89
2.30E-40 1467392_T subsp. trichocarpa Ceres CLONE Artificial
Sequence 291 95.89 1.80E-40 ID no. 25162_T Ceres Annot: Populus
balsamifera 292 95.89 1.40E-40 1529647_T subsp. trichocarpa Ceres
CLONE Artificial Sequence 293 95.89 1.40E-40 ID no. 1405728_T
Public GI no. Artificial Sequence 294 95.38 1.70E-35 54288726_T
Public GI no. Artificial Sequence 295 95.1 2.10E-55 50906823_T
Public GI no. Artificial Sequence 296 95.1 5.50E-55 83306206_T
Public GI no. Artificial Sequence 297 95.1 3.40E-55 20152203_T
Public GI no. Artificial Sequence 298 94.52 3.80E-40 40287568_T
Public GI no. Artificial Sequence 299 94.12 7.00E-55 50929483_T
Ceres Annot: Populus balsamifera 300 93.15 7.80E-40 1450556_T
subsp. trichocarpa Ceres CLONE Artificial Sequence 301 93.14
1.50E-54 ID no. 1031152_T Public GI no. Artificial Sequence 302
93.14 1.50E-54 52548244_T Public GI no. Artificial Sequence 303
92.86 1.30E-37 82621144_T Ceres CLONE Artificial Sequence 304 92.16
4.90E-54 ID no. 878043_T Public GI no. Artificial Sequence 305
92.16 4.90E-54 34909292_T Public GI no. Artificial Sequence 306
92.16 4.90E-54 2668744_T Ceres CLONE Artificial Sequence 307 91.78
1.60E-39 ID no. 511132_T Ceres Annot: Populus balsamifera 308 91.78
2.10E-39 1533930_T subsp. trichocarpa Ceres Annot: Populus
balsamifera 309 91.14 1.80E-42 1495171_T subsp. trichocarpa Public
GI no. Artificial Sequence 310 94.12 7.00E-55 20086317_T Ceres
CLONE Artificial Sequence 311 90.41 7.00E-39 ID no. 10022_T Ceres
CLONE Artificial Sequence 312 90.41 7.00E-39 ID no. 12547_T Ceres
CLONE Artificial Sequence 313 90.41 7.00E-39 ID no. 27679_T Public
GI no. Artificial Sequence 314 90.2 1.70E-53 20259611_T Public GI
no. Artificial Sequence 315 90.2 9.20E-53
30025160_T Public GI no. Artificial Sequence 316 89.22 1.70E-53
50904839_T Ceres CLONE Artificial Sequence 317 89.22 1.50E-52 ID
no. 1063753_T Ceres Annot: Populus balsamifera 318 89.04 1.90E-38
1533218_T subsp. trichocarpa Public GI no. Artificial Sequence 319
88.89 4.90E-38 297880_T Ceres CLONE Artificial Sequence 320 88.24
1.90E-52 ID no. 1357060_T Public GI no. Artificial Sequence 321
87.25 5.10E-52 54402104_T Public GI no. Artificial Sequence 322
87.25 2.20E-51 50725323_T Ceres CLONE Artificial Sequence 323 87.25
1.70E-51 ID no. 376667_T Ceres CLONE Artificial Sequence 324 86.81
2.70E-46 ID no. 256705_T Public GI no. Artificial Sequence 325
86.27 8.30E-52 20259629_T Public GI no. Artificial Sequence 326
86.27 1.10E-51 52851174_T Ceres CLONE Artificial Sequence 327 86.27
2.80E-51 ID no. 1061097_T Public GI no. Artificial Sequence 328
86.27 7.70E-49 1373001_T Public GI no. Artificial Sequence 329
86.27 1.70E-51 66354468_T Public GI no. Artificial Sequence 330
82.35 4.10E-50 4100646_T
TABLE-US-00032 TABLE 32 Percent identity to Ceres Clone 36412 (SEQ
ID NO: 332) SEQ ID % Iden- Designation Species NO: tity e-value
Public GI no. Arabidopsis thaliana 333 98.5 2.00E-176 3152583 Ceres
Annot: Populus balsamifera 335 60.2 1.20E-68 1467033_PRT subsp.
trichocarpa Ceres Annot: Populus balsamifera 337 60.1 7.90E-70
1536919_PRT subsp. trichocarpa Ceres CLONE Glycine max 338 52.7
1.90E-61 ID no. 1641329 Ceres CLONE Glycine max 339 52 1.39E-65 ID
no. 1650419 Ceres CLONE Glycine max 340 51 1.09E-63 ID no.
597699
TABLE-US-00033 TABLE 33 Percent identity to Ceres Clone 4829 (SEQ
ID NO: 346) SEQ ID % Iden- Designation Species NO: tity e-value
Ceres CLONE Arabidopsis thaliana 347 87.2 8.89E-62 ID no. 24885
Ceres CLONE Arabidopsis thaliana 348 84.2 1.19E-57 ID no. 27878
Ceres Annot: Populus balsamifera 350 81.2 1.50E-52 1485102_PRT
subsp. trichocarpa Ceres CLONE Glycine max 351 72.8 2.30E-54 ID no.
1646533 Public GI no. Oryza sativa subsp. 352 63.4 5.99E-40
29371519 japonica Public GI no. Oryza sativa subsp. 353 63.4
5.99E-40 38347602 japonica Public GI no. Ipomoea trifida 354 60.8
1.29E-39 45935148 Ceres CLONE Zea mays 355 60.4 3.79E-38 ID no.
359934 Ceres CLONE Zea mays 356 58.8 5.99E-40 ID no. 294598 Ceres
CLONE Triticum aestivum 357 55.1 3.10E-36 ID no. 839270 Ceres CLONE
Gossypium hirsutum 525 78.6 1.40E-54 ID no. 1836904 Ceres CLONE
Gossypium hirsutum 526 77.3 7.99E-54 ID no. 1932013 Ceres CLONE
Panicum virgatum 527 58.1 7.79E-40 ID no. 1768109
TABLE-US-00034 TABLE 34 Percent identity to Ceres Clone 5426 (SEQ
ID NO: 359) SEQ ID % Iden- Designation Species NO: tity e-value
Ceres CLONE Brassica napus 360 90.3 3.99E-59 ID no. 1123542 Ceres
CLONE Arabidopsis thaliana 361 90.2 4.80E-63 ID no. 9083 Public GI
no. Arabidopsis thaliana 362 90.2 4.80E-63 79322493 Ceres CLONE
Arabidopsis thaliana 363 90.2 4.80E-63 ID no. 265408 Ceres CLONE
Arabidopsis thaliana 364 90.2 4.80E-63 ID no. 32164 Ceres CLONE
Brassica napus 365 88.1 2.39E-61 ID no. 1068047 Ceres CLONE
Brassica napus 366 87.4 6.30E-61 ID no. 965035 Ceres Annot: Populus
balsamifera 368 76.7 1.00E-53 1499194_PRT subsp. trichocarpa Ceres
Annot: Populus balsamifera 370 75.5 3.40E-53 1439584_PRT subsp.
trichocarpa Ceres CLONE Glycine max 371 73.4 5.60E-53 ID no. 557065
Ceres CLONE Glycine max 372 72.5 1.29E-51 ID no. 465060 Ceres CLONE
Zea mays 512 87.4 2.99E-61 ID no. 1458107 Ceres CLONE Gossypium
hirsutum 513 76.2 1.10E-54 ID no. 1932511 Ceres CLONE Gossypium
hirsutum 514 75.5 7.99E-54 ID no. 1850967 Ceres CLONE Gossypium
hirsutum 515 75.5 1.00E-53 ID no. 1835707 Ceres CLONE Musa
acuminata 516 72.7 5.19E-50 ID no. 1727213 Ceres CLONE Panicum
virgatum 517 71.3 9.69E-49 ID no. 1767577 Ceres CLONE Musa
acuminata 518 71.3 8.50E-50 ID no. 1712104 Ceres CLONE Panicum
virgatum 519 71.1 1.20E-48 ID no. 1778377 Public GI no. Solanum
tuberosum 520 70.6 4.10E-50 76573317 Ceres CLONE Panicum virgatum
521 69.9 1.29E-46 ID no. 1787980 Public GI no. Oryza sativa subsp.
522 69.7 6.89E-48 115465181 japonica Public GI no. Oryza sativa
subsp. 523 69.7 8.69E-48 48716267 japonica Ceres CLONE Triticum
aestivum 524 66.1 3.39E-46 ID no. 575833
TABLE-US-00035 TABLE 35 Percent identity to Ceres Clone 7894 (SEQ
ID NO: 374) SEQ ID % Iden- Designation Species NO: tity e-value
Public GI no. Brassica oleracea 375 94.6 6.89E-261 18091781 Public
GI no. Ricinus communis 376 75.3 2.39E-194 468562 Ceres Annot:
Populus balsamifera 378 74.7 4.30E-188 1479767_PRT subsp.
trichocarpa Public GI no. Euphorbia esula 379 74.3 9.99E-194
7649151 Ceres Annot: Populus balsamifera 381 72.5 3.70E-182
1486712_PRT subsp. trichocarpa Public GI no. Glycine max 382 71.9
3.29E-181 33620334 Public GI no. Solanum tuberosum 383 71.6
4.39E-179 439294 Public GI no. Populus tremula .times. 384 71.6
8.90E-181 77153413 Populus tremuloides Public GI no. Pisum sativum
385 71.1 1.40E-180 5230818 Public GI no. Alonsoa meridionalis 386
70.9 1.29E-179 17447420 Public GI no. Vicia faba 387 70.8 4.39E-179
1935019 Public GI no. Juglans regia 388 69.6 8.79E-174 51863031
Public GI no. Nicotiana tabacum 389 69.3 4.39E-179 575351 Public GI
no. Vitis vinifera 390 68.7 7.39E-177 68161544 Public GI no. Vitis
vinifera 391 68.5 1.49E-176 6434833 Public GI no. Spinacia oleracea
392 68.2 9.99E-173 21319 Public GI no. Beta vulgaris 393 67.7
2.69E-170 5823000 Public GI no. Beta vulgaris subsp. 394 67.3
8.99E-172 633172 vulgaris Public GI no. Asarina barclaiana 395 67.2
5.50E-172 6120115 Public GI no. Plantago major 396 66.2 7.39E-177
415988 Public GI no. Hevea brasiliensis 528 73 9.09E-188 116008246
Ceres CLONE Gossypium hirsutum 529 66.8 4.09E-176 ID no.
1925996
Example 21
Transgenic Plants Containing Homologs and/or Orthologs
[0412] Cloned sequences of some of the functional homologs and/or
orthologs of protein-modulating polypeptides that were identified
as outlined in Example 20 were used to make transgenic plants.
[0413] Ceres Clone 39130 (SEQ ID NO:440) is a cDNA clone isolated
from Arabidopsis that encodes a functional homologue of SEQ ID
NO:193, and is predicted to encode a 119 amino acid transcription
ubiquitin-conjugating enzyme polypeptide. Ceres Clone 424395 (SEQ
ID NO:427) is a cDNA clone isolated from Zea mays that encodes a
functional homologue of SEQ ID NO:136, and is predicted to encode a
446 amino acid tryptophan/tyrosine permease family polypeptide.
Ceres Clone 24885 (SEQ ID NO:460) is a cDNA clone isolated from
Arabidopsis that encodes a functional homologue of SEQ ID NO:346,
and is predicted to encode a 156 amino acid polypeptide having a
DUF662 domain. Ceres Clone 300692 (SEQ ID NO:418) is a cDNA clone
isolated from Zea mays that encodes a functional homologue of SEQ
ID NO:111, and is predicted to encode an 87 amino acid
cyclin-dependent kinase regulatory subunit polypeptide. Ceres Clone
944737 (SEQ ID NO:422) is a cDNA clone isolated from Brassica napus
that encodes a functional homologue of SEQ ID NO:111, and is
predicted to encode an 81 amino acid cyclin-dependent kinase
regulatory subunit polypeptide.
[0414] A construct was made with the CRS 311 vector that contained
Ceres Clone 39130 operably linked to the 32449 promoter. Constructs
were made with the CRS 338 vector that contained Ceres Clone
424395, Ceres Clone 24885, Ceres Clone 300692, or Ceres Clone
944737, each operably linked to a CaMV 35S promoter. Wild-type
Arabidopsis thaliana ecotype Wassilewskija (Ws) plants were
transformed separately with each construct as described in Example
1.
[0415] Transgenic Arabidopsis lines containing Ceres Clone 39130,
Ceres Clone 424395, Ceres Clone 24885, Ceres Clone 300692, or Ceres
Clone 944737 were designated ME00177, ME06339, ME07409, ME08044, or
ME08488, respectively. The presence of each vector containing a
Ceres clone described above in the respective transgenic
Arabidopsis line transformed with the vector was confirmed by
Finale.TM. resistance, polymerase chain reaction (PCR)
amplification from green leaf tissue extract, and/or sequencing of
PCR products. As controls, wild-type Arabidopsis ecotype Ws plants
were transformed with the empty vector CRS 338 or the empty vector
CRS 311.
Example 22
Results for Transgenic Plants Containing Homologs and/or
Orthologs
[0416] T.sub.2 seed from three events of ME00177 containing Ceres
Clone 39130 was analyzed for total oil content using FT-NIR
spectroscopy as described in Example 2.
[0417] The oil content in T.sub.2 seed from three events of ME00177
was increased compared to the mean oil content in seed from
transgenic Arabidopsis lines planted within 30 days of ME00177. As
presented in Table 36, the oil content was increased to 115%, 109%,
and 122% in seed from events -01, -02, and -03, respectively,
compared to the population mean.
TABLE-US-00036 TABLE 36 Oil content (% control) in T.sub.2 seed
from ME00177 events containing Ceres Clone 39130 Event -01 Event
-02 Event -03 Control Protein 115 109 122 100 .+-. 7* content (%
control) in T.sub.2 seed p-value** 0.04 0.15 <0.01 N/A
*Population mean of the protein content in seed from transgenic
lines planted within 30 days of ME00177. Variation is presented as
standard error of the mean. **The p-values for T.sub.2 seed were
calculated using z-scores.
[0418] T.sub.2 seed from three events of ME06339 containing Ceres
Clone 424395 was analyzed for total oil content using FT-NIR
spectroscopy as described in Example 2.
[0419] The oil content in T.sub.2 seed from three events of ME06339
was increased compared to the mean oil content in seed from
transgenic Arabidopsis lines planted within 30 days of ME06339. As
presented in Table 37, the protein content was increased to 110%,
103%, and 110% in seed from events -02, -03, and -04, respectively,
compared to the population mean.
TABLE-US-00037 TABLE 37 Oil content (% control) in T.sub.2 seed
from ME06339 events containing Ceres Clone 424395 Event -02 Event
-03 Event -04 Control Protein 110 103 110 100 .+-. 12* content (%
control) in T.sub.2 seed p-value 0.06 0.401 0.068 N/A *Population
mean of the protein content in seed from transgenic lines planted
within 30 days of ME06339. Variation is presented as standard error
of the mean.
[0420] T.sub.2 seed from four events of ME07409 containing Ceres
Clone 24885 was analyzed for total oil content using FT-NIR
spectroscopy as described in Example 2.
[0421] The oil content in T.sub.2 seed from four events of ME07409
was modulated compared to the mean oil content in seed from
transgenic Arabidopsis lines planted within 30 days of ME07409. As
presented in Table 38, the oil content was increased to 107%, 103%,
and 108% in seed from events -02, -03, and -04, respectively,
compared to the population mean, while the oil content was
decreased to 98% of the population mean in event -06.
TABLE-US-00038 TABLE 38 Oil content (% control) in T.sub.2 seed
from ME07409 events containing Ceres Clone 24885 Event -02 Event
-03 Event -04 Event -06 Control Protein 107 103 108 108 100 .+-.
13* content (% control) in T.sub.2 seed p-value** 0.06 0.224 0.04
0.551 N/A *Population mean of the protein content in seed from
transgenic lines planted within 30 days of ME07409. Variation is
presented as standard error of the mean. **The p-values for T.sub.2
seed were calculated using z-scores.
[0422] T.sub.2 seed from four events of ME08044 containing Ceres
Clone 300692 was analyzed for total oil content using FT-NIR
spectroscopy as described in Example 2.
[0423] The oil content in T.sub.2 seed from four events of ME08044
was modulated compared to the mean oil content in seed from
transgenic Arabidopsis lines planted within 30 days of ME08044. As
presented in Table 39, the protein content was increased to 108%
and 105% in seed from events -01 and -02, respectively, compared to
the population mean, while the oil content was decreased to 95% and
88% of the population mean in events -03 and -04, respectively.
TABLE-US-00039 TABLE 39 Oil content (% control) in T.sub.2 seed
from ME08044 events containing Ceres Clone 300692 Event -01 Event
-02 Event -03 Event -04 Control Protein 108 105 95 88 100 .+-. 15*
content (% control) in T.sub.2 seed p-value** 0.094 0.215 0.112
<0.01 N/A *Population mean of the protein content in seed from
transgenic lines planted within 30 days of ME08044. Variation is
presented as standard error of the mean. **The p-values for T.sub.2
seed were calculated using z-scores.
[0424] T.sub.2 seed from five events of ME08488 containing Ceres
Clone 944737 was analyzed for total oil content using FT-NIR
spectroscopy as described in Example 2.
[0425] The oil content in T.sub.2 seed from five events of ME08488
was modulated compared to the mean oil content in seed from
transgenic Arabidopsis lines planted within 30 days of ME08488. As
presented in Table 40, the protein content was increased to 103%
and 107% in seed from events -02 and -04, respectively, compared to
the population mean, while the oil content was decreased to 97%,
84%, and 90% of the population mean in events -01, -03, and -05,
respectively.
TABLE-US-00040 TABLE 40 Oil content (% control) in T.sub.2 seed
from ME08488 events containing Ceres Clone 944737 Event Event -01
Event -02 Event -03 Event -04 -05 Control Protein 97 103 84 107 90
100 .+-. 18* content (% control) in T.sub.2 seed p-value** 0.48
0.248 0.104 0.148 0.746 N/A *Population mean of the protein content
in seed from transgenic lines planted within 30 days of ME08488.
Variation is presented as standard error of the mean. **The
p-values for T.sub.2 seed were calculated using z-scores.
[0426] Transgenic plants containing cloned sequences of some of the
other functional homologs and/or orthologs of Example 21 were
analyzed for total oil content in seeds by FT-NIR spectroscopy. The
results were inconclusive.
Other Embodiments
[0427] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120331583A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120331583A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References