U.S. patent application number 13/154797 was filed with the patent office on 2011-11-03 for increase in yield by reducing gene expression.
This patent application is currently assigned to Metanomics GmbH. Invention is credited to Agnes Chardonnens, Piotr Puzio.
Application Number | 20110271402 13/154797 |
Document ID | / |
Family ID | 35589098 |
Filed Date | 2011-11-03 |
United States Patent
Application |
20110271402 |
Kind Code |
A1 |
Puzio; Piotr ; et
al. |
November 3, 2011 |
INCREASE IN YIELD BY REDUCING GENE EXPRESSION
Abstract
The present invention relates to a process for the increase in
yield in a plant organism by reducing gene expression. The
invention furthermore relates to nucleic acid molecules,
polypeptides, nucleic acid constructs, vectors, antisense
molecules, antibodies, host cells, plant tissue, propagation
material, harvested material and plants.
Inventors: |
Puzio; Piotr; (Mariakerke
(gent), BE) ; Chardonnens; Agnes; (Nh Enkhuizen,
NL) |
Assignee: |
Metanomics GmbH
Berlin
DE
|
Family ID: |
35589098 |
Appl. No.: |
13/154797 |
Filed: |
June 7, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11791706 |
May 25, 2007 |
7982095 |
|
|
PCT/EP2005/012650 |
Nov 26, 2005 |
|
|
|
13154797 |
|
|
|
|
Current U.S.
Class: |
800/286 ;
800/285 |
Current CPC
Class: |
C12N 15/8261 20130101;
Y02A 40/146 20180101 |
Class at
Publication: |
800/286 ;
800/285 |
International
Class: |
A01H 1/00 20060101
A01H001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 27, 2004 |
EP |
04028287.3 |
Claims
1. A process for the increase in yield of a plant, which comprises
reducing or deleting the expression of a protein in a plant,
wherein the reducing or deleting comprises: a) introducing into a
plant a nucleic acid molecule encoding a ribonucleic acid sequence,
which is able to form a double-stranded ribonucleic acid molecule,
whereby the sense strand of said double-stranded ribonucleic acid
molecule (i) has a homology of at least 80% to a nucleic acid
sequence encoding the protein of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID
NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID
NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,
SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID
NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,
SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID
NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO:
104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO:
112; or (ii) has a homology of at least 80% to a nucleic acid
sequence encoding the protein of SEQ ID NO: 114, SEQ ID NO: 116,
SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ
ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID
NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288; or (iii) comprises a fragment of at least 20,
21, 22, 23, 24 or 25 bases of the nucleic acid sequence of (i) or
(ii) above; and whereby the antisense strand of the double-stranded
ribonucleic acid molecule is complementary to said sense strand; b)
introducing into a plant an antisense nucleic acid molecule,
whereby the antisense nucleic acid molecule is antisense to the
full length of the transcribed mRNA of a protein encoded by a
nucleic acid sequence selected from the group consisting of i) a
nucleic acid sequence encoding the polypeptide of SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ
ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:
32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ
ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:
50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ
ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:
68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ
ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:
86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110,
or SEQ ID NO: 112; ii) the nucleic acid sequence of SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ
ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:
31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ
ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ
ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO:
67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ
ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:
85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109,
or SEQ ID NO: 111; iii) a nucleic acid sequence encoding a
polypeptide comprising an amino acid sequence having at least 80%
identity to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8,
SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID
NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or
SEQ ID NO: 112, having the biological activity of an OPT
oligopeptide transporter, and comprising the sequence of SEQ ID NO:
88, SEQ ID NO: 89, and SEQ ID NO: 90; iv) a nucleic acid sequence
encoding the protein of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO:
286, or SEQ ID NO: 288; v) the nucleic acid sequence of SEQ ID NO:
113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287; and vi) a
nucleic acid sequence encoding a polypeptide comprising an amino
acid sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID
NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, or SEQ ID NO:
288, having the biological activity of an ABC transporter, and
comprising the sequence of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID
NO: 267, and/or SEQ ID NO: 268; (c) introducing into a plant a
ribozyme which specifically cleaves a nucleic acid molecule
conferring expression of a protein having the biological activity
of a protein encoded by a nucleic acid sequence selected from the
group consisting of i) a nucleic acid sequence encoding the
polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:
28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ
ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ
ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:
64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ
ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:
82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106,
SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112; ii) the nucleic
acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,
SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID
NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,
SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID
NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,
SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID
NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71,
SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID
NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO:
105, SEQ ID NO: 107, SEQ ID NO: 109, or SEQ ID NO: 111; iii) a
nucleic acid sequence encoding a polypeptide comprising an amino
acid sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:
16, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ
ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 58, SEQ ID NO:
62, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112, having the
biological activity of an OPT oligopeptide transporter, and
comprising the sequence of SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID
NO: 90; iv) a nucleic acid sequence encoding the protein of SEQ ID
NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286, or SEQ ID NO: 288; v) the
nucleic acid sequence of SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:
117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO:
125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO:
133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO:
141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO:
149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:
157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO:
165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:
173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:
181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO:
189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO:
197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO:
205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO:
213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO:
221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO:
229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO:
237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO:
245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO:
269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO:
277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO:
285, or SEQ ID NO: 287; and vi) a nucleic acid sequence encoding a
polypeptide comprising an amino acid sequence having at least 80%
identity to the amino acid sequence of SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288, having the biological
activity of an ABC transporter, and comprising the sequence of SEQ
ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, and/or SEQ ID NO: 268;
d) introducing into a plant the antisense nucleic acid molecule
characterized in (b) and the ribozyme characterized in (c); e)
introducing into a plant a sense nucleic acid molecule conferring
the expression of a nucleic acid sequence comprising i) a nucleic
acid sequence encoding the polypeptide of SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ
ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:
42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ
ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:
60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ
ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:
78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ
ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ
ID NO: 112; ii) the nucleic acid sequence of SEQ ID NO: 3, SEQ ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID
NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID
NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID
NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, or
SEQ ID NO: 111; iii) a nucleic acid sequence encoding a polypeptide
comprising an amino acid sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 42, SEQ ID NO: 44, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 64,
SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID
NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 106, SEQ ID NO:
108, SEQ ID NO: 110, or SEQ ID NO: 112, having the biological
activity of an OPT oligopeptide transporter, and comprising the
sequence of SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90; iv) a
nucleic acid sequence encoding the protein of SEQ ID NO: 114, SEQ
ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID
NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286, or SEQ ID NO: 288; v) the nucleic acid
sequence of SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID
NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO:
287; and vi) a nucleic acid sequence encoding a polypeptide
comprising an amino acid sequence having at least 80% identity to
the amino acid sequence of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID
NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO:
286, or SEQ ID NO: 288, having the biological activity of an ABC
transporter, and comprising the sequence of SEQ ID NO: 265, SEQ ID
NO: 266, SEQ ID NO: 267, and/or SEQ ID NO: 268; for inducing
co-suppression of an endogenous oligopeptide transporter protein;
or f) introducing into a plant an expression construct conferring
the expression of any one of (a) to (e); and expressing said
nucleic acid molecule, wherein said expression results in increased
plant yield.
2. The process of claim 1, wherein the plant is selected from the
group consisting of Anacardiaceae, Asteraceae, Apiaceae,
Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae,
Cannabaceae, Convolvulaceae, Chenopodiaceae, Cucurbitaceae,
Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae,
Gramineae, Juglandaceae, Lauraceae, Leguminosae, Linaceae,
perennial grass, a fodder crop, a vegetable, and an ornamental.
3. The process of claim 1, wherein the nucleic acid sequence of b)
iii), c) iii), and e) iii) encodes a polypeptide comprising an
amino acid sequence having at least 90% identity to the amino acid
sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:
16, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ
ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 58, SEQ ID NO:
62, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112, has the
biological activity of an OPT oligopeptide transporter protein 2,
and comprises the sequence of SEQ ID NO: 88, SEQ ID NO: 89, and SEQ
ID NO: 90; or wherein the nucleic acid sequence of b) vi), c) vi),
and e) vi) encodes a polypeptide comprising an amino acid sequence
having at least 90% identity to the amino acid sequence of SEQ ID
NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286, or SEQ ID NO: 288, having the
biological activity of an ABC transporter, and comprising the
sequence of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, and/or
SEQ ID NO: 268.
4. The process of claim 1, wherein the nucleic acid sequence of b)
iii), c) iii), and e) iii) encodes a polypeptide comprising an
amino acid sequence having at least 95% identity to the amino acid
sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO:
16, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ
ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 58, SEQ ID NO:
62, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112, has the
biological activity of an OPT oligopeptide transporter protein 2,
and comprises the sequence of SEQ ID NO: 88, SEQ ID NO: 89, and SEQ
ID NO: 90; or wherein the nucleic acid sequence of b) vi), c) vi),
and e) vi) encodes a polypeptide comprising an amino acid sequence
having at least 95% identity to the amino acid sequence of SEQ ID
NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286, or SEQ ID NO: 288, having the
biological activity of an ABC transporter, and comprising the
sequence of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, and/or
SEQ ID NO: 268.
5. The process of claim 1, wherein the plant is selected from the
group consisting of maize, soya, canola, wheat, barley, triticale,
rice, linseed, sunflower, potato, and Arabidopsis.
6. The process of claim 1, wherein the reducing or deleting
comprises introducing into a plant a nucleic acid molecule encoding
a ribonucleic acid sequence, which is able to form a
double-stranded ribonucleic acid molecule, whereby the sense strand
of said double-stranded ribonucleic acid molecule (i) has a
homology of at least 80% to a nucleic acid sequence encoding the
protein of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID
NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,
SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID
NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46,
SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID
NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64,
SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID
NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,
SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ
ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112; or (ii) has a
homology of at least 80% to a nucleic acid sequence encoding the
protein of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID
NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; or (iii) comprises a fragment of at least 20, 21, 22, 23, 24
or 25 bases of the nucleic acid sequence of (i) or (ii) above; and
whereby the antisense strand of the double-stranded ribonucleic
acid molecule is complementary to said sense strand.
7. The process of claim 1, wherein the reducing or deleting
comprises introducing into a plant an antisense nucleic acid
molecule, whereby the antisense nucleic molecule is antisense to
the full length of the transcribed mRNA of a protein encoded by a
nucleic acid sequence selected from the group consisting of i) a
nucleic acid sequence encoding the polypeptide of SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ
ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:
32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ
ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:
50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ
ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:
68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ
ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:
86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110,
or SEQ ID NO: 112; ii) the nucleic acid sequence of SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ
ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:
31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ
ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ
ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO:
67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ
ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:
85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109,
or SEQ ID NO: 111; iii) a nucleic acid sequence encoding a
polypeptide comprising an amino acid sequence having at least 80%
identity to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8,
SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID
NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or
SEQ ID NO: 112, having the biological activity of an OPT
oligopeptide transporter, and comprising the sequence of SEQ ID NO:
88, SEQ ID NO: 89, and SEQ ID NO: 90; iv) a nucleic acid sequence
encoding the protein of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO:
286, or SEQ ID NO: 288; v) the nucleic acid sequence of SEQ ID NO:
113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287; and vi) a
nucleic acid sequence encoding a polypeptide comprising an amino
acid sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID
NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288, having the biological activity of an ABC transporter, and
comprising the sequence of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID
NO: 267, and/or SEQ ID NO: 268.
8. The process of claim 1, wherein the reducing or deleting
comprises introducing into a plant a ribozyme which specifically
cleaves a nucleic acid molecule conferring expression of a protein
having the biological activity of a protein encoded by a nucleic
acid sequence selected from the group consisting of i) a nucleic
acid sequence encoding the polypeptide of SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ
ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:
42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ
ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:
60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ
ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:
78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ
ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ
ID NO: 112; ii) the nucleic acid sequence of SEQ ID NO: 3, SEQ ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID
NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID
NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID
NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, or
SEQ ID NO: 111; iii) a nucleic acid sequence encoding a polypeptide
comprising an amino acid sequence having at least 80% identity to
the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO:
12, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ
ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
58, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID NO: 72, SEQ
ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:
82, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO:
112, having the biological activity of an OPT oligopeptide
transporter, and comprising the sequence of SEQ ID NO: 88, SEQ ID
NO: 89, and SEQ ID NO: 90; iv) a nucleic acid sequence encoding the
protein of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID
NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286, or SEQ ID NO:
288; v) the nucleic acid sequence of SEQ ID NO: 113, SEQ ID NO:
115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:
123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:
187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:
195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:
203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:
211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:
219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:
227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:
235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:
243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO:
251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO:
275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO:
283, SEQ ID NO: 285, or SEQ ID NO: 287; and vi) a nucleic acid
sequence encoding a polypeptide comprising an amino acid sequence
having at least 80% identity to the amino acid sequence of SEQ ID
NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288, having the
biological activity of an ABC transporter, and comprising the
sequence of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, and/or
SEQ ID NO: 268.
9. The process of claim 1, wherein the reducing or deleting
comprises introducing into a plant the antisense nucleic acid
molecule characterized in (b) and the ribozyme characterized in
(c).
10. The process of claim 1, wherein the reducing or deleting
comprises introducing into a plant a sense nucleic acid molecule
conferring the expression of a nucleic acid sequence comprising i)
a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, or SEQ ID NO: 112; ii) the nucleic acid sequence of SEQ ID NO:
3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,
SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID
NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57,
SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID
NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75,
SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID
NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO:
109, or SEQ ID NO: 111; iii) a nucleic acid sequence encoding a
polypeptide comprising an amino acid sequence having at least 80%
identity to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8,
SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID
NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or
SEQ ID NO: 112, having the biological activity of an OPT
oligopeptide transporter, and comprising the sequence of SEQ ID NO:
88, SEQ ID NO: 89, and SEQ ID NO: 90; iv) a nucleic acid sequence
encoding the protein of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO:
286, or SEQ ID NO: 288; v) the nucleic acid sequence of SEQ ID NO:
113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287; and vi) a
nucleic acid sequence encoding a polypeptide comprising an amino
acid sequence having at least 80% identity to the amino acid
sequence of SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID
NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288, having the biological activity of an ABC transporter, and
comprising the sequence of SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID
NO: 267, and/or SEQ ID NO: 268.
11. The process of claim 1, wherein the nucleic acid sequence
comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID
NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,
SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,
SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID
NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID
NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,
SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID
NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, or SEQ ID NO: 111; or
wherein the nucleic acid sequence encodes a polypeptide comprising
the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:
44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ
ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:
62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ
ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:
80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104,
SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, or SEQ ID NO: 112.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/791,706 filed May 25, 2007, which is a
national stage application (under 35 U.S.C. .sctn.371) of
PCT/EP2005/012650 filed Nov. 26, 2005, which claims benefit to
European application 0 402 8287.3 filed Nov. 27, 2004. The entire
contents of each of these applications are hereby incorporated by
reference herein in their entirety.
SUBMISSION OF SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is
Sequence_List.sub.--13195.sub.--00027_US. The size of the text file
is 1387 KB, and the text file was created on Jun. 2, 2011.
DESCRIPTION
[0003] The present invention relates to a process for the increase
in yield in plants by reduction or deletion of the biological
activity represented by a protein as depicted in SEQ ID NO: 2, SEQ
ID NO: 113 or its homologs and growing the plant under conditions
which permit increased plant growth.
[0004] The invention furthermore relates to a nucleic acid molecule
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ
ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:
27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ
ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ
ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:
63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ
ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:
81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105,
SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ
ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID
NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:
187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:
195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:
203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:
211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:
219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:
227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:
235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:
243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO:
251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO:
275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO:
283, SEQ ID NO: 285 or SEQ ID NO: 287
[0005] and a polypeptide SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 and nucleic acid constructs, vectors, antisense molecules,
antibodies, host cells, plant tissue, propagation material,
harvested material, plants as well as agricultural compositions and
to their use.
[0006] Ever since useful plants were first cultivated, increasing
the crop yield has, in addition to improving resistance to abiotic
and biotic stress, been the most important aim when growing new
plant varieties. Means as diverse as tilling, fertilizing,
irrigation, cultivation or crop protection agents, to name but a
few, are used for improving yields. Thus, cultivation successes in
increasing the crop, for example by increasing the seed setting,
and those in reducing the loss of crop, for example owing to bad
weather, i.e. weather which is too dry, too wet, too hot or too
cold, or due to infestation with pests such as, for example,
insects, fungi or bacteria, complement one another. In view of the
rapidly growing world population, a substantial increase in yield,
without extending the economically arable areas, is absolutely
necessary in order to provide sufficient food and, at the same
time, protect other existing natural spaces.
[0007] The methods of classical genetics and cultivation for
developing new varieties with better yields are increasingly
supplemented by genetic methods. Thus, genes have been identified
which are responsible for particular properties such as resistance
to abiotic or biotic stress or growth rate control. Interesting
genes or gene products thereof may be appropriately regulated in
the desired useful plants, for example by mutation,
(over)expression or reduction/inhibition of such genes or their
products, in order to achieve the desired increased yield or higher
tolerance to stress.
[0008] The same applies to microorganisms and useful animals, the
breeding of which is primarily and especially concerned with
likewise achieving a particular biomass or a particular weight more
rapidly, in addition to higher resistance to biotic or abiotic
stress.
[0009] One example of a strategy resulting in better or more rapid
plant growth is to increase the photosynthetic capability of plants
(U.S. Pat. No. 6,239,332 and DE 19940270). This approach, however,
is promising only if the photosynthetic performance of said plants
is growth-limiting. Another approach is to modulate regulation of
plant growth by influencing cell cycle control (WO 01/31041, CA
2263067, WO 00/56905, WO 00/37645). However, a change in the
plant's architecture may be the undesired side effect of a massive
intervention in the control of plant growth (WO 01/31041; CA
2263067). Other approaches may involve putative transcriptional
regulators as for example claimed in WO 02/079403 or US
2003/013228. Such transcriptional regulators often occur in gene
families, in which the family members might display significant
cross talk and/or antagonistic control. In addition the function of
transcription factors rely on the precise presence of their
recognition sequences in the target organisms. This fact might
complicate the transfer of result from model species to target
organisms.
[0010] Despite a few very promising approaches, there is
nevertheless still a great need of providing methods for preparing
organisms with faster growth and higher yield, expecially by gene
reduction methods, which do not involve the expression of a
heterologous transgene.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 depicts the vector 1bxPcUbicolic.
[0012] FIG. 2 depicts the vector 10xPeUbiSpacer.
DETAILED DESCRIPTION
[0013] It was now found that this object is achieved by providing
the process according to the invention described herein and the
embodiments characterized in the claims.
[0014] Accordingly, in a first embodiment, the invention relates to
a process for the increase in seed yield and freshweight of plants.
Accordingly, in the present invention, the term "yield" as used
herein relates to "increase in fresh weight of seed or plant
material".
[0015] In one embodiment, the term "yield" means dry matter of seed
and plant material.
[0016] Accordingly, the invention relates to a process for the
increase in yield, which comprises the following steps: [0017] a)
reduction or deletion of the biological activity represented by the
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 in a plant, and [0018] b) growing the plant under conditions
which permit the increase in yield in said plant.
[0019] Advantageously a genetic modification of the plant leads to
an enhanced yield of seed weight and/or fresh weight in the plant.
The terms "enhanced" or "increase" mean at least a 5%, 10%, 15%,
20% or 25%, preferably at least 30%, 40%, 50%, 60% or 70%, more
preferably 80%, 90%, 100%, 150% or 200% higher production in yield
in comparison to the reference as defined below, that means in
comparison to the plant without the aforementioned modification of
the biological activity of protein of the invention.
[0020] Preferably, this process further includes the step of
harvesting the seed, which is produced by the plant.
[0021] Surprisingly, the transgenic reduction or deletion of the
expression of the protein of the invention in Arabidopsis thaliana
conferred an increase in yield as measured as an increase in seed
weight and fresh weight of the transformed plants.
[0022] In accordance with the invention, the term "plant" as
understood herein relates to a monocot or a dicot plant, the whole
plant, the seed, plant parts as tubers, roots, leaves or cell(s)
thereof.
[0023] In accordance with the invention, a protein or polypeptide
has the "activity or preferably biological activity" of the protein
as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:
44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ
ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:
62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ
ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:
80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104,
SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ
ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID
NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 if in the
event its de novo activity or biological activity is reduced or
deleted leads to an increase in yield. That means the reduction or
deletion of its biological activity for example its enzymatic
activity is somehow related to the increase in yield. Throughout
the specification the reduction or deletion of the biological
activity of such a aforementioned protein or polypeptide or a
nucleic acid molecule or sequence encoding such protein or
polypeptide means a reduction of its biological activity for
example its enzymatic activity of at least 10% preferably 20%, 30%,
40% or 50%, particularly preferably 60% 70% or 80%, most
particularly preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% in comparison to the biological activity of the
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO; 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288. Throughout the specification a deletion of the biological
activity of the protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288 means a total loss of the activity. The reduction
or deletion of the biological activity leads to an increase of the
seed weight or the fresh weight of at least 10%, 20%, 30%, 40%,
50%, 100%, 150% or 200%, preferably of at least 250% or 300%,
particularly preferably of at least 350% or 400%, most particularly
preferably of at least 500% or 600% or more.
[0024] The terms "reduction", "decrease" or "deletion" relate to a
corresponding change of a property in a plant, a plant part such as
a tissue, seed, root, leaves, flower etc. or in a cell. Under
"change of a property" it is understood that the activity,
expression level or amount of a gene product or the metabolite
content is changed in a specific volume or in a specific amount of
protein relative to a corresponding volume or amount of protein of
a control, reference or wild type. Preferably, the overall activity
in the volume is reduced, decreased or deleted in cases if the
reduction, decrease or deletion is related to the reduction,
decrease or deletion of an activity of a gene product, independent
whether the amount of gene product or the specific activity of the
gene product or both is reduced, decreased or deleted or whether
the amount, stability or translation efficacy of the nucleic acid
sequence or gene encoding for the gene product is reduced,
decreased or deleted.
[0025] The terms "reduction", "decrease" or "deletion" include the
change of said property in only parts of the subject of the present
invention, for example, the modification can be found in
compartment of a cell, like an organelle, or in a part of a plant,
like tissue, seed, root, leaves, flowers etc. but is detectable if
the overall subject, i.e. complete cell or plant, is tested.
Preferably, the "reduction", "decrease" or "deletion" is found
cellular, thus the term "reduction, decrease or deletion of an
activity" or "reduction, decrease or deletion of a metabolite
content" relates to the cellular reduction, decrease or deletion
compared to the wild type cell. In addition the terms "reduction",
"decrease" or "deletion" include the change of said property only
during different growth phases of the organism used in the
inventive process, for example the reduction, decrease or deletion
takes place only during the seed growth or during blooming.
Furthermore the terms include a transitional reduction, decrease or
deletion for example because the used RNAi is not stable integrated
in the genome of the organism and has therefore only a transient
effect or is for example controlled by an inducible promoter.
[0026] Accordingly, the term "reduction", "decrease" or "deletion"
means that the specific activity of an enzyme or a protein or
regulatory RNA as well as the amount of a compound or metabolite,
e.g. of a polypeptide, a nucleic acid molecule or the fine chemical
of the invention or an encoding mRNA or DNA, can be reduced,
decreased or deleted in a volume.
[0027] The terms "wild type", "control" or "reference" are
exchangeable and can be a cell or a part of the plant such as an
organelle or tissue, which was not modified or treated according to
the herein described process according to the invention.
Accordingly, the cell or a part of plant such as an organelle or a
tissue, used as wild type, control or reference corresponds to the
cell, organism or part thereof as much as possible and is in any
other property but in the result of the process of the invention as
identical to the subject matter of the invention as possible. Thus,
the wild type, control or reference is treated identically or as
identical as possible, saying that only conditions or properties
might be different which do not influence the quality of the tested
property.
[0028] Preferably, any comparison is carried out under analogous
conditions. The term "analogous conditions" means that all
conditions such as, for example, culture or growing conditions,
assay conditions (such as buffer composition, temperature,
substrates, pathogen strain, concentrations and the like) are kept
identical between the experiments to be compared.
[0029] The "reference", "control", or "wild type" is preferably a
subject, e.g. an organelle, a cell, a tissue, an organism, in
particular a plant or a microorganism, which was not modified or
treated according to the herein described process of the invention
and is in any other property as similar to the subject matter of
the invention as possible. The reference, control or wild type is
in its genome, transcriptome, proteome or metabolome as similar as
possible to the subject of the present invention. Preferably, the
term "reference-" "control-" or "wild type-"-organelle, -cell,
-tissue or -plant relates to a plant which is nearly genetically
identical to the organelle, cell, tissue or plant, of the present
invention or a part thereof preferably 95%, more preferred are 98%,
even more preferred are 99.00%, in particular 99.10%, 99.30%,
99.50%, 99.70%, 99.90%, 99.99%, 99.999% or more. Most preferable
the "reference", "control", or "wild type" is preferably a subject,
e.g. an organelle, a cell, a tissue, an organism, which is
genetically identical to the organism, cell organelle used
according to the process of the invention except that nucleic acid
molecules or the gene product encoded by them are changed according
to the inventive process.
[0030] Preferably, the reference, control or wild type differs form
the subject of the present invention only in the cellular activity
of the polypeptide or RNA of the invention, e.g. as result of a
reduction, decrease or deletion in the level of the nucleic acid
molecule of the present invention or a reduction, decrease or
deletion of the specific activity of the polypeptide or RNA of the
invention, e.g. by the expression level or activity of protein or
RNA that means its biological activity and/or its biochemical or
genetic causes.
[0031] The term "expression" means the transcription of a gene into
structural RNA (rRNA, tRNA, miRNA) or messenger RNA (mRNA) with the
subsequent translation of the latter into a protein.
Experimentally, expression can be detected by e.g. Northern, qRT
PCR, transcriptional run-on assays or Western blotting and other
immuno assays. As consequence of the reduction, decrease or
deletion of the expression that means as consequence of the
reduced, decreased or deleted transcription of a gene a related
phenotypic trait appears such as the enhanced or increased
production of the fine chemical.
[0032] Accordingly, preferred reference subject is the starting
subject of the present process of the invention. Preferably, the
reference and the subject matter of the invention are compared
after standardization and normalization, e.g. to the amount of
total RNA, DNA, or Protein or activity or expression of reference
genes, like housekeeping genes, such as ubiquitin.
[0033] A series of mechanisms exists via which a modification in
the polypeptide of the invention can directly or indirectly affect
the yield, production and/or production efficiency of the amino
acid. For example, the molecule number or the specific activity of
the polypeptide of the invention or the number of expression of the
nucleic acid molecule of the invention may be reduced, decreased or
deleted. However, it is also possible to reduce, decrease or delete
the expression of the gene which is naturally present in the
organisms, for example by modifying the regulation of the gene, or
by reducing or decreasing the stability of the mRNA or of the gene
product encoded by the nucleic acid molecule of the invention.
[0034] This also applies analogously to the combined reduction,
decrease or deletion of the expression of the nucleic acid molecule
of the present invention or its gene product together with the
manipulation of additional activities such as e.g. biosynthetic
enzymes.
[0035] The reduction, decrease, deletion or modulation according to
this invention can be constitutive, e.g. due to a stable permanent
transgenic expression or to a stable mutation in the corresponding
endogenous gene encoding the nucleic acid molecule of the invention
or to a modulation of the expression or of the behaviour of a gene
conferring the expression of the polypeptide of the invention, or
transient, e.g. due to an transient transformation, a transiently
active promotor or temporary addition of a modulator such as an
antagonist or inductor, e.g. after transformation with a inducible
construct carrying a double-stranded RNA nucleic acid molecule, an
antisense nucleic acid molecule, a ribozyme of the invention etc.
under control of a inducible promoter and adding the inducer, e.g.
tetracycline or as described herein below.
[0036] The reduction, decrease or deletion in activity amounts
preferably by at least 10%, preferably by at least 30% or at least
60%, especially preferably by at least 70%, 80%, 85%, 90% or more,
very especially preferably are at least 95%, more preferably are at
least 99% or more in comparison to the control, reference or wild
type. Most preferably the reduction, decrease or deletion in
activity amounts to 100%.
[0037] The specific activity of a polypeptide encoded by a nucleic
acid molecule of the present invention or of the polypeptide of the
present invention can be tested as described in the examples. In
particular, the reduction, decrease or deletion of the expression
of a protein in question in a cell, e.g. a plant cell or a
microorganism and the detection of an increase of the fine chemical
level as well as in comparison to a control is an easy test and can
be performed as described in the examples.
[0038] The term "reduction", "decrease" or "deletion" includes,
that the reason for said "reduction", "decrease" or "deletion is a
chemical compound, which is administered to the organism.
[0039] Accordingly, in the following, the term "reducing",
"decreasing" or "deleting" also comprises the term "debasing",
"depleting", diminishing" or "bringing down". The decreased or
reduced activity manifests itself in increased yield. In this
context, the yield, is increased by 3% or more, especially
preferably are 10% or more, very especially preferably are more
than 30% and most preferably are 70% or more, such as 100%, 300% or
500% or more.
[0040] A protein having a biological activity of the proteins used
in the inventive process preferably has the structure of the
polypeptide described herein, in particular of the polypeptides
shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 or the
functional homologues thereof as described herein, or is encoded by
the nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID
NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,
SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID
NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID
NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:
111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287
or its herein described functional homologues and has the
abovementioned activity.
[0041] For the purposes of the present invention, the terms "yield"
also encompass the corresponding plant parts, such as, for example
leaves, seed, roots or stem. Preferably the term yield is intended
to encompass the term seed fresh weight, seed dry weight, plant
fresh weight, plant dry weight, leaf number, leaf fresh weight,
leaf dry weight, root fresh weight, root dry weight, number of
blossoms, number of leaves, fruit fresh weight and fruit dry
weight, blossom fresh weight, blossom dry weight.
[0042] The term "expression" refers to the transcription and/or
translation of a codogenic gene segment or gene. As a rule, the
resulting product is a mRNA or a protein. However, expression
products can also include functional RNAs such as, for example,
antisense, tRNAs, snRNAs, rRNAs, dsRNA, siRNA, miRNAs, ribozymes
etc. Expression may be systemic, local or temporal, for example
limited to certain cell types, tissues, organs or time periods.
[0043] In one embodiment, the process of the present invention
comprises one or more of the following steps [0044] a)
destabilizing a protein enabling the reduced, decreased or deleted
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the biological activity of a protein as depicted
in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,
SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID
NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,
SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID
NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,
SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID
NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,
SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID
NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO:
106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 leading to
the herein-mentioned yield increasing activity; or [0045] b)
destabilizing a mRNA enabling the reduced, decreased or deleted
expression of a protein encoded by the nucleic acid molecule of the
invention, e.g. of a polypeptide having the biological activity of
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 or of a mRNA encoding the polypeptide of the present invention
leading to the herein-mentioned yield increasing activity; or
[0046] c) Increasing the biological activity of a protein or RNA
e.g. increasing the biological activity of a repressor enabling the
reduced, decreased or deleted expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention leading to the herein-mentioned yield
increasing activity, e.g. of a polypeptide having the biological
activity of the protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288 or increasing the inhibitory regulation of the
polypeptide of the invention; or [0047] d) Increasing the
biological activity of a protein or RNA e.g. increasing the
biological activity of a repressor enabling the reduced, decreased
or deleted expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention leading to the herein-mentioned yield increasing
activity, e.g. of a polypeptide having the biological activity of
the protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ
ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:
42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ
ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:
60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ
ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:
78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ
ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID
NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 by adding one or more exogenous repression factors such as a
chemical compound for example an inhibitor to the plant or parts
thereof; or [0048] e) reducing, decreasing or deleting the copy
number of a gene e.g. reducing, decreasing or deleting the copy
number of a gene encoding an activator enabling the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned yield
increasing activity, e.g. of a polypeptide having the biological
activity of the protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288 or [0049] f) reducing, decreasing or deleting the
expression of the endogenous gene encoding the polypeptide of the
invention, e.g. a polypeptide having the biological activity of the
protein used in the inventive process such as the protein as
depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 by adding for
example an antisense molecule or RNAi or improving the activity of
negative expression elements. This can be achieved for example
through the expression of the nucleic acids of the invention or
parts of it in antisense orientation or by the expression of
hairpin RNAi constructs or the simultaneous expression of sense and
antisense RNA for the nucleic acids of the invention. Details are
described later in the description or in the examples; or
[0050] g) Increasing the biological activity of a protein or RNA
leading to a dominant negative phenotype of the biological activity
of the nucleic acid molecule of the invention or the protein of the
invention such as a protein as depicted in SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288. Advantageously this can be achieved for example
through the expression of the nucleic acids encoding a protein,
which has lost its biological activity and which binds to another
protein in a multimeric complex and thereby decreasing or deleting
the activity of said complex or which binds for example as a
transcription factor to DNA and thereby decreasing or deleting the
activity of the translated protein; or [0051] h) expression of an
antibody or aptamer, which binds to the nucleic acid molecule of
the invention or the protein of the invention such as a protein as
depicted SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 and thereby
reducing, decreasing or deleting its biological activity; and/or
[0052] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the nucleic acid
molecule encoding the protein of the invention or the protein
itself is reduced, decreased or deleted. This can be achieved by
e.g. modulating light and/or nutrient conditions, which in terms
modulated the expression of the gene or protein of the
invention.
[0053] Preferably, said mRNA is one kind of the nucleic acid
molecule of the present invention and/or the protein enabling the
reduced or decreased expression of a protein encoded by the nucleic
acid molecule of the present invention or the polypeptide having
the herein mentioned biological activity of the polypeptide of the
present invention, e.g. conferring the increase in yield after
decreasing the expression or activity of the encoded polypeptide or
having the biological activity of a polypeptide having the
biological activity of the protein of the invention.
[0054] In general, the amount of mRNA, polynucleotide or nucleic
acid molecule in a cell or a compartment of an organism correlates
to the amount of encoded protein and thus with the overall activity
of the encoded protein in said volume. Said correlation is not
always linear, the activity in the volume is dependent on the
stability of the molecules, the degradation of the molecules or the
presence of activating or inhibiting co-factors. Further, product
and metabolite inhibitions of enzymes are well known.
[0055] The activity of the abovementioned protein and/or
polypeptide encoded by the nucleic acid molecule of the present
invention can be reduced, decreased or deleted in various ways. For
example, the activity in an organism or in a part thereof, like a
cell, is reduced or decreased via reducing or decreasing the gene
product number, e.g. by reducing or decreasing the expression rate,
like mutating the natural promoter to a lower activity, or by
reducing or decreasing the stability of the mRNA expressed, thus
reducing or decreasing the translation rate, and/or reducing or
decreasing the stability of the gene product, thus increasing the
proteins decay. Further, the activity or turnover of enzymes or
channels or carriers, transcription factors, and similar active
proteins can be influenced in such a manner that a reduction of the
reaction rate or a modification (reduction, decrease or deletion)
of the affinity to the substrate results, is reached. A mutation in
the catalytic centre of an polypeptide of the invention, e.g. as
enzyme, can modulate the turn over rate of the enzyme, e.g. a knock
out of an essential amino acid can lead to a reduced or complete
knock out of the activity of the enzyme, or the deletion of
regulator binding sites can reduce a negative regulation like a
feedback inhibition (or a substrate inhibition, if the substrate
level is also increased). The specific activity of an transporter
of the present invention can be decreased such that the transport
rate is decreased. Reducing the stability of the encoding mRNA or
the protein can also decrease the activity of a gene product. The
reduction of the activity is also under the scope of the term
"reduced, decreased or deleted activity". Beside this,
advantagously the reduction of the activity in cis, e.g. mutating
the promotor including other cis-regulatory elements, or the
transcribed or coding parts of the gene, inhibition can be achieved
in trans, e.g. by transfactors like chimeric transcription factor,
ribozymes, antisense RNAs, dsRNAs antibodies or dominant negative
proteins versions, which interfere with various stages of
expression, e.g. the transcription, the translation or the activity
of the protein or protein complex itself.
[0056] In the inventive process as mentioned above preferably the
reduction, decrease or deletion of the biological activity
represented by protein of the invention is achieved by reducing,
decreasing or deleting the expression of at least one nucleic acid
molecule, wherein the nucleic acid molecule is selected from the
group consisting of: [0057] a) nucleic acid molecule encoding the
polypeptide shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ
ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:
16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ
ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:
34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ
ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO:
52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ
ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:
70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ
ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO:
104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; [0058] b) nucleic acid molecule comprising the nucleic acid
molecule shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID
NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID
NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID
NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,
SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID
NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:
103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:
111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287; [0059] c) nucleic acid molecule comprising a nucleic acid
sequence, which, as a result of the degeneracy of the genetic code,
can be derived from a polypeptide sequence depicted in SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,
SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID
NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,
SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID
NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,
SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID
NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,
SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID
NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO:
108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288; [0060] d) nucleic acid
molecule encoding a polypeptide having at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and having the biological activity
represented by protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288; [0061] e) nucleic acid molecule which comprises
a polynucleotide which is obtained by amplifying a cDNA library or
a genomic library using the primers depicted in SEQ ID NO:92 and
SEQ ID NO: 93 or 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: 264, SEQ ID
NO: 265 and having the biological activity represented by proteins
as depicted in SEQ ID NO: 2 or SEQ ID NO: 113; [0062] f) nucleic
acid molecule encoding a polypeptide which is isolated with the aid
of monoclonal or polyclonal antibodies against a polypeptide
encoded by one of the nucleic acid molecules of (a) to (d or e) and
having the biological activity represented by the protein as
depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288; [0063] g)
nucleic acid molecule encoding a polypeptide comprising the
consensus sequence shown in SEQ ID NO: 87 or SEQ ID NO: 88 or SEQ
ID NO: 89 or SEQ ID NO: 90 or SEQ ID NO: 91 or SEQ ID NO: 265 or
SEQ ID NO: 266 or SEQ ID NO: 267 or SEQ ID NO: 268 and having the
biological activity represented by the protein as depicted in SEQ
ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID
NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,
SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID
NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46,
SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID
NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64,
SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID
NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,
SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ
ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID
NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 28; [0064] h) nucleic acid
molecule encoding a polypeptide having the biological activity
represented by the protein as depicted in SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288;
[0065] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridisation
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) or (b) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(c) and encoding a polypeptide having the biological activity
represented by protein of the invention
[0066] or which comprises a sequence which is complementary
thereto.
[0067] Moreover, the regulation of the abovementioned nucleic acid
sequences may be modified so that gene expression is decreased.
This reduction, decrease or deletion (reduction, decrease,
deletion, inactivation or down-regulation shall be used as synonyms
throughout the specification) can be achieved as mentioned above by
all methods known to the skilled person, preferably by
double-stranded RNA interference (dsRNAi), introduction of an
antisense nucleic acid, a ribozyme, an antisense nucleic acid
combined with a ribozyme, a nucleic acid encoding a co-suppressor,
a nucleic acid encoding a dominant negative protein, DNA- or
protein-binding factors targeting said gene or -RNA or -proteins,
RNA degradation inducing viral nucleic acids and expression
systems, systems for inducing a homologous recombination of said
genes, mutations in said genes or a combination of the above.
[0068] In general, an activity of a gene product in a plant or part
thereof, in particular in a plant cell or a plant tissue can be
decreased by decreasing the amount of the specific encoding mRNA or
the corresponding protein in said plant or part thereof. "amount of
protein or mRNA" is understood as meaning the molecule number of
polypeptides or mRNA molecules in a plant, a plant tissue, a plant
cell or a cell compartment. "Decrease" in the amount of a protein
means the quantitative decrease of the molecule number of said
protein in a plant, a plant tissue, a plant cell or a cell
compartment--for example by one of the methods described herein
below--in comparison to a wild type, control or reference.
[0069] In this context, inactivation means that the enzymatic or
biological activity of the polypeptides encoded is no longer
detectable in the plant or in the plant cell. For the purposes of
the invention, down regulation (=reduction) means that the
enzymatic or biological activity of the polypeptides encoded is
partly or completely reduced in comparison with the activity of the
untreated plant. This can be achieved by different cell-biological
mechanisms. In this context, the activity can be down regulated in
the entire plant or in individual parts of the plant, for example
in tissues such as the seed, the leaf, the root or other parts. In
this context, the enzymatic activity or biological activity is
reduced by at least 10%, advantageously at least 20%, preferably at
least 30%, especially preferably at least 40%, 50% or 60%, very
especially preferably at least 70%, 80%, 85% or 90% or more, very
especially preferably are at least 95%, more preferably are at
least 99% or more in comparison to the control, reference or wild
type. Most preferably the reduction, decrease or deletion in
activity amounts to 100%.
[0070] Various strategies for reducing the quantity, the
expression, the activity or the function of proteins encoded by the
nucleic acids or the nucleic acid sequences itself according to the
invention are encompassed in accordance with the invention. The
skilled worker will recognize that a series of different methods
are available for influencing the quantity of a protein, the
activity or the function in the desired manner.
[0071] The term "biological activity" means the biological function
of the protein of the invention. In contrast to the term
"biological activity" the term "activity" means the increase in
yield produced by the inventive process. The term "biological
activity" preferably refers to for example the enzymatic function,
transporter or carrier function, DNA-packaging function, heat shock
protein function, recombination protein function or regulatory
function of a peptide or protein in a plant, a plant tissue, a
plant cell or a cell compartment. Suitable substrates are
low-molecular-weight compounds and also the protein interaction
partners of a protein. The term "reduction" of the biological
function refers, for example, to the quantitative reduction in
binding capacity or binding strength of a protein for at least one
substrate in a plant, a plant tissue, a plant cell or a cell
compartment--for example by one of the methods described herein
below--in comparison with the wild type of the same genus and
species to which this method has not been applied, under otherwise
identical conditions (such as, for example, culture conditions, age
of the plants and the like). Reduction is also understood as
meaning the modification of the substrate specificity as can be
expressed for example, by the kcat/Km value. In this context, a
reduction of the function of at least 10%, advantageously of at
least 20%, preferably at least 30%, especially preferably of at
least 40%, 50% or 60%, very especially preferably of at least 70%,
80%, 90% or 95%, in comparison with the untreated plant is
advantageous. A particularly advantageous embodiment is the
inactivation of the function, e.g. the function of a transporter
membrane protein. Binding partners for the protein can be
identified in the manner with which the skilled worker is familiar,
for example by the yeast 2-hybrid system.
[0072] A modification, i.e. a decrease, can be caused by endogenous
or exogenous factors. For example, a decrease in activity in a
plant or a part thereof can be caused by adding a chemical compound
such as an antagonist to the media, nutrition, soil of the plants
or to the plants themselves.
[0073] In one embodiment the increase in yield can be achieved by
decreasing the endogenous level of the polypeptide of the
invention. Accordingly, in an embodiment of the present invention,
the present invention relates to a process, wherein the gene copy
number of a gene encoding the polynucleotide or nucleic acid
molecule of the invention is decreased. Further, the endogenous
level of the polypeptide of the invention can for example be
decreased by modifiying the transcriptional or translational
regulation of the polypeptide.
[0074] A further embodiment of the inventive process is a process,
whereby the reduction or deletion of the biological activity
represented by the protein used in the inventive process is
achieved by a process comprising a step selected from the group
consisting of: [0075] (a) introducing of a nucleic acid molecule
encoding a ribonucleic acid sequence, which are able to form
double-stranded ribonucleic acid molecules, whereby the sense
strand of said double-stranded ribonucleic acid molecules has a
homology of at least 30% to a nucleic acid molecule conferring the
expression of or encoding a protein having the biological activity
of the protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID
NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,
SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID
NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,
SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID
NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID
NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,
SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID
NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86,
SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ
ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID
NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 or comprising a fragment of at least 17, 18, 19, 20, 21, 22,
23, 24, or 25 base pairs of a nucleic acid molecule with a homology
of at least 50% to a nucleic acid molecule conferring the
expression of a protein having the biological activity of the
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; [0076] (b) introducing an antisense nucleic acid molecule,
whereby the antisense nucleic acid molecule has a homology of at
least 30% to a nucleic acid molecule antisense to a nucleic acid
molecule encoding a protein having the biological activity of the
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; or conferring the expression of a protein having the
biological activity of a protein having the biological activity of
the protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ
ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:
42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ
ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO:
60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ
ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO:
78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ
ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID
NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; or introducing an antisense nucleic acid molecule comprising a
fragment of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25
base pairs of a nucleic acid molecule with a homology of at least
50% to an antisense nucleic acid molecule to a nucleic acid
molecule conferring the expression of a protein having the
biological activity of protein as depicted in SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID
NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,
SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID
NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,
SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID
NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84,
SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ
ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID
NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288; [0077] (c) introducing of a ribozyme which
specifically cleaves a nucleic acid molecule conferring expression
of a protein having the biological activity of protein as depicted
in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,
SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID
NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,
SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID
NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,
SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID
NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,
SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID
NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO:
106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288; [0078] (d)
introducing of the antisense nucleic acid molecule characterized in
(b) or the ribozyme characterized in (c); [0079] (e) introducing of
a sense nucleic acid molecule conferring the expression of a
nucleic acid molecule as depicted in SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ
ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:
31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ
ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ
ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO:
67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ
ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83 or SEQ ID
NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO:
109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:
117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO:
125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO:
133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO:
141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO:
149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:
157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO:
165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:
173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:
181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO:
189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO:
197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO:
205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO:
213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO:
221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO:
229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO:
237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO:
245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO:
269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO:
277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285
or SEQ ID NO: 287 or a nucleic acid molecule encoding a polypeptide
or part of it (need not to encode a functional protein) having at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of used in the inventive
process and having the biological activity represented by the
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ
ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID
NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 for inducing
a co-suppression of the endogenous protein having a biological
activity of the protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288;
[0080] (f) introducing a nucleic acid molecule conferring the
expression of a dominant-negative mutant of a protein having the
biological activity of a protein as depicted in SEQ ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID
NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,
SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID
NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,
SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID
NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84,
SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ
ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID
NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288 that means by expressing said sequence leading to
the dominant-negative mutant protein thereby the biological
activity of the protein used in the inventive process is reduced,
decreased or deleted and therefore the yield is increased; [0081]
(g) introducing a nucleic acid molecule encoding a factor, which
binds to a nucleic acid molecule conferring the expression of a
protein having the biological activity of a protein as depicted in
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:
28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ
ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ
ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:
64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ
ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:
82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106,
SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ
ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID
NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288; [0082] (h) introducing a
viral nucleic acid molecule conferring the decline of a RNA
molecule conferring the expression of a protein having the
biological activity of a protein used in the inventive process
especially a protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ
ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:
32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ
ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:
50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ
ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:
68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ
ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:
86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110,
SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ
ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID
NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; [0083] (i) introducing a nucleic acid construct capable to
recombine with a endogenous gene conferring the expression of a
protein having the biological activity of a protein used in the
inventive process especially a protein as depicted in SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ
ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:
48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ
ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108,
SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ
ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID
NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288; [0084] (j) introducing a non-silent mutation in
an endogenous gene conferring the expression of a protein having
the biological activity of a protein used in the inventive process
especially a protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ
ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:
32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ
ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:
50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ
ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:
68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ
ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:
86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110,
SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ
ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID
NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; [0085] (k) selecting of a non-silent mutation in a nucleic
acid sequence encoding a protein having the biological activity of
a protein used in the inventive process especially a protein as
depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 from a
randomly mutagenized population of organisms used in the inventive
process; and/or [0086] (l) introducing an expression construct
conferring the expression of nucleic acid molecule characterized in
any one of (a) to (k).
[0087] As the skilled person knows it is possible starting from the
nucleic acid sequences mentioned under point (a) to (j) above it is
easy possible to isolate the 5'- and/or 3'-regions of said nucleic
acid sequences and to use said 5'- and/or 3'-sequences for the
reduction, decrease or deletion of the nucleic acid sequences used
in the inventive process according to the different process steps
(a) to (j) mentioned above. Preferably less than 1000 bp, 900 bp,
800 bp or 700 bp, particular preferably less than 600 bp, 500 bp,
400 bp, 300 bp, 200 bp or 100 bp of the 5'- and/or 3'-region of the
said nucleic acid sequence are used.
[0088] The aforementioned process steps of the reduction or
deletion of the biological activity represented by the protein of
the invention lead to an increase in yield.
[0089] A reduction in the activity or the function is preferably
achieved by a reduced expression of a gene encoding the protein of
the inventive process.
[0090] Further preferred embodiments of the invention to reduce the
biological activity of the protein of the inventive process, said
reduction of the activity or function can be achieved using the
following methods: [0091] a) introduction of a double-stranded RNA
nucleic acid sequence (dsRNA) as described above or of an
expression cassette, or more than one expression cassette, ensuring
the expression of the latter; [0092] b) introduction of an
antisense nucleic acid sequence or of an expression cassette
ensuring the expression of the latter. Encompassed are those
methods in which the antisense nucleic acid sequence is directed
against a gene (i.e. genomic DNA sequences) or a gene transcript
(i.e. RNA sequences) including the 5' and 3' non-translated
regions. Also encompassed are .alpha.-anomeric nucleic acid
sequences; [0093] c) introduction of an antisense nucleic acid
sequence in combination with a ribozyme or of an expression
cassette ensuring the expression of the former; [0094] d)
introduction of sense nucleic acid sequences for inducing
cosuppression or of an expression cassette ensuring the expression
of the former; [0095] e) introduction of a nucleic acid sequence
encoding dominant-negative protein or of an expression cassette
ensuring the expression of the latter; [0096] f) introduction of
DNA-, RNA- or protein-binding factors against genes, RNA's or
proteins or of an expression cassette ensuring the expression of
the latter; [0097] g) introduction of viral nucleic acid sequences
and expression constructs which bring about the degradation of RNA,
or of an expression cassette ensuring the expression of the former;
[0098] h) introduction of constructs for inducing homologous
recombination on endogenous genes, for example for generating
knockout mutants; [0099] i) introduction of mutations into
endogenous genes for generating a loss of function (e.g. generation
of stop codons, reading-frame shifts and the like); and/or [0100]
j) identifying a non silent mutation e.g. generation of stop
codons, reading-frame shifts, inversions and the like in random
mutagenized population according to the so called tilling method.
[0101] k) introduction of constructs for expression of an antibody
which specifically binds and thereby inactivates the polypeptides
of the invention.
[0102] Each of these methods may bring about a reduction in the
expression, the activity or the function for the purposes of the
invention. A combined use is also feasible. Further methods are
known to the skilled worker and may encompass hindering or
preventing processing of the protein, transport of the protein or
its mRNA, inhibition of ribosomal attachment, inhibition of RNA
splicing, induction of an enzyme which degrades RNA or the protein
of the invention and/or inhibition of translational elongation or
termination.
[0103] What follows is a brief description of the individual
preferred methods:
A) Introduction of a Double-Stranded RNA Nucleic Acid Sequence
(dsRNA)
[0104] The method of regulating genes by means of double-stranded
RNA ("double-stranded RNA interference"; dsRNAi) has been described
extensively for animal, yeast, fungi and plant organisms such as
Neurospora, Zebrafish, Drosophila, mice, planaria, humans,
Trypanosoma, petunia or Arabidopsis (for example Matzke M A et al.
(2000) Plant Mol. Biol. 43: 401-415; Fire A. et al. (1998) Nature
391: 806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914;
WO 00/44895; WO 00/49035; WO 00/63364). In addition RNAi is also
documented as an advantageously tool for the repression of genes in
bacteria such as E. coli for example by Tchurikov et al. [J. Biol.
Chem., 2000, 275 (34): 26523-26529]. Fire et al. named the
phenomenon RNAi for RNA interference. The techniques and methods
described in the above references are expressly referred to.
Efficient gene suppression can also be observed in the case of
transient expression or following transient transformation, for
example as the consequence of a biolistic transformation (Schweizer
P et al. (2000) Plant J 2000 24: 895-903). dsRNAi methods are based
on the phenomenon that the simultaneous introduction of
complementary strand and counterstrand of a gene transcript brings
about highly effective suppression of the expression of the gene in
question. The resulting phenotype is very similar to that of an
analogous knock-out mutant (Waterhouse P M et al. (1998) Proc.
Natl. Acad. Sci. USA 95: 13959-64).
[0105] Tuschl et al. [Gens Dev., 1999, 13 (24): 3191-3197] was able
to show that the efficiency of the RNAi method is a function of the
length of the duplex, the length of the 3'-end overhangs, and the
sequence in these overhangs. Based on the work of Tuschl et al. and
assuming that the underlining principles are conserved between
different species the following guidelines can be given to the
skilled worker: [0106] to achieve good results the 5' and 3'
untranslated regions of the used nucleic acid sequence and regions
close to the start codon should be in general avoided as this
regions are richer in regulatory protein binding sites and
interactions between RNAi sequences and such regulatory proteins
might lead to undesired interactions; [0107] in plants the 5' and
3' untranslated regions of the used nucleic acid sequence and
regions close to the start codon preferably 50 to 100 nt upstream
of the start codon give good results and therefore should not be
avoided; [0108] preferably a region of the used mRNA is selected,
which is 50 to 100 nt (=nucleotides or bases) downstream of the AUG
start codon; [0109] only dsRNA (=double-stranded RNA) sequences
from exons are useful for the method, as sequences from introns
have no effect; [0110] the G/C content in this region should be
greater than 30% and less than 70% ideally around 50%; [0111] a
possible secondary structure of the target mRNA is less important
for the effect of the RNAi method.
[0112] The dsRNAi method has proved to be particularly effective
and advantageous for reducing the expression of the nucleic acid
sequences of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID
NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID
NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID
NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,
SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID
NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:
103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:
111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 and/or homologs thereof. As described inter alia in WO
99/32619, dsRNAi approaches are clearly superior to traditional
antisense approaches.
[0113] The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about
increased yield activity by the reduction in the expression of the
nucleic acid sequences of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID
NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID
NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID
NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ
ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID
NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 and/or homologs thereof. In a double-stranded RNA molecule for
reducing the expression of an protein encoded by a nucleic acid
sequence of one of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID
NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,
SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID
NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID
NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:
111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 and/or homologs thereof, [0114] i) one of the two RNA strands
is essentially identical to at least part of a nucleic acid
sequence, and [0115] the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[0116] The term "essentially identical" refers to the fact that the
dsRNA sequence may also include insertions, deletions and
individual point mutations in comparison to the target sequence
while still bringing about an effective reduction in expression.
Preferably, the homology as defined above amounts to at least 30%,
preferably at least 40%, 50%, 60%, 70% or 80%, very especially
preferably at least 90%, most preferably 100%, between the "sense"
strand of an inhibitory dsRNA and a part-segment of a nucleic acid
sequence of the invention including in a preferred embodiment of
the invention their endogenous 5'- and 3' untranslated regions or
between the "antisense" strand and the complementary strand of a
nucleic acid sequence, respectively. The part-segment amounts to at
least 10 bases, preferably at least 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29 or 30 bases, especially preferably at least 40,
50, 60, 70, 80 or 90 bases, very especially preferably at least
100, 200, 300 or 400 bases, most preferably at least 500, 600, 700,
800, 900 or more bases or at least 1000 or 2000 bases or more in
length. In another preferred embodiment of the invention the
part-segment amounts to 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or
27 bases, preferably to 20, 21, 22, 23, 24 or 25 bases. These short
sequences are preferred in plants. The longer sequences preferably
between 200 and 800 bases are also useable in plants. Long
double-stranded RNAs are processed in the plant into many siRNAs
small/short interfering RNAs) for example by the protein Dicer,
which is a ds-specific Rnase III enzyme. As an alternative, an
"essentially identical" dsRNA may also be defined as a nucleic acid
sequence, which is capable of hybridizing with part of a gene
transcript (for example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM
EDTA at 50.degree. C. or 70.degree. C. for 12 to 16 h).
[0117] The dsRNA may consist of one or more strands of polymerized
ribonucleotides. Modification of both the sugar-phosphate backbone
and of the nucleosides may furthermore be present. For example, the
phosphodiester bonds of the natural RNA can be modified in such a
way that they encompass at least one nitrogen or sulfur heteroatom.
Bases may undergo modification in such a way that the activity of,
for example, adenosine deaminase is restricted. These and other
modifications are described herein below in the methods for
stabilizing antisense RNA.
[0118] The dsRNA can be prepared enzymatically; it may also be
synthesized chemically, either in full or in part. Short dsRNA up
to 30 bp, which effectively mediate RNA interference, can be for
example efficiently generated by partial digestion of long dsRNA
templates using E. coli ribonuclease III (RNase III). (Yang, D., et
al. (2002) Proc. Natl. Acad. Sci. USA 99, 9942.)
[0119] The double-stranded structure can be formed starting from a
single, self-complementary strand or starting from two
complementary strands. In a single, self-complementary strand,
"sense" and "antisense" sequence can be linked by a linking
sequence ("linker") and form for example a hairpin structure.
Preferably, the linking sequence may take the form of an intron,
which is spliced out following dsRNA synthesis. The nucleic acid
sequence encoding a dsRNA may contain further elements such as, for
example, transcription termination signals or polyadenylation
signals. If the two strands of the dsRNA are to be combined in a
cell or a plant, this can be brought about in a variety of ways:
[0120] a) transformation of the cell or of the plant, with a vector
encompassing the two expression cassettes; [0121] b)
cotransformation of the cell or of the plant, with two vectors, one
of which encompasses the expression cassettes with the "sense"
strand while the other encompasses the expression cassettes with
the "antisense" strand; [0122] c) supertransformation of the cell
or of the plant, with a vector encompassing the expression
cassettes with the "sense" strand, after the cell or the plant had
already been trans-formed with a vector encompassing the expression
cassettes with the "antisense" strand; [0123] d) hybridization e.g.
crossing of two organisms, advantageously of plants, each of which
has been transformed with one vector, one of which encompasses the
expression cassettes with the "sense" strand while the other
encompasses the expression cassettes with the "antisense" strand;
[0124] e) introduction of a construct comprising two promoters that
lead to transcription of the desired sequence from both directions;
and/or [0125] f) infecting of the cell or of the plant with an
engineered virus, which is able to produce the desired dsRNA
molecule.
[0126] Formation of the RNA duplex can be initiated either outside
the cell or within the cell. If the dsRNA is synthesized outside
the target cell or plant it can be introduced into the plant or a
cell of the plant by injection, microinjection, electroporation,
high velocity particles, by laser beam or mediated by chemical
compounds (DEAE-dextran, calciumphosphate, liposomes).
[0127] As shown in WO 99/53050, the dsRNA may also encompass a
hairpin structure, by linking the "sense" and "antisense" strands
by a "linker" (for example an intron). The self-complementary dsRNA
structures are preferred since they merely require the expression
of a construct and always encompass the complementary strands in an
equimolar ratio.
[0128] The expression cassettes encoding the "antisense" or the
"sense" strand of the dsRNA or the self-complementary strand of the
dsRNA are preferably inserted into a vector and stably inserted
into the genome of a plant, using the methods described herein
below (for example using selection markers), in order to ensure
permanent expression of the dsRNA. Transient expression with
bacterial or viral vectors are similar useful.
[0129] The dsRNA can be introduced using an amount which makes
possible at least one copy per cell. A larger amount (for example
at least 5, 10, 100, 500 or 1 000 copies per cell) may bring about
more efficient reduction.
[0130] As has already been described, 100% sequence identity
between the dsRNA and a gene transcript of a nucleic acid sequence
of one of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ
ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:
25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ
ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:
61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ
ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO:
79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103,
SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ
ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID
NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287 or it's
homolog is not necessarily required in order to bring about
effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence of one
of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,
SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID
NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,
SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID
NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,
SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID
NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71,
SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID
NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO:
105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO:
113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO; 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287 or homologs
thereof of one plant, may be used to suppress the corresponding
expression in another plant.
[0131] Due to the high degree of sequence homology between SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ
ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:
19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ
ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:
37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ
ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:
55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ
ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:
73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ
ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID
NO: 107, SEQ ID NO: 109, SEQ ID NO: 111 and SEQ ID NO: 113, SEQ ID
NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:
123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:
187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:
195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:
203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:
211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:
219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:
227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:
235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:
243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO:
251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO:
275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO:
283, SEQ ID NO: 285 or SEQ ID NO: 287 respectively from various
organisms (e.g. plants), allows the conclusion that these proteins
may be conserved to a high degree within, for example other plants,
it is optionally possible so that the expression of a dsRNA derived
from one of the disclosed sequences as shown in SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID
NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,
SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID
NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65,
SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID
NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83,
SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ
ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID
NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO:
125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO:
133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO:
141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO:
149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:
157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO:
165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:
173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:
181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO:
189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO:
197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO:
205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO:
213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO:
221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO:
229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO:
237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO:
245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO:
269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO:
277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285
or SEQ ID NO: 287 or homologs thereof should also have an
advantageous effect in other plant species.
[0132] The dsRNA can be synthesized either in vivo or in vitro. To
this end, a DNA sequence encoding a dsRNA can be introduced into an
expression cassette under the control of at least one genetic
control element (such as, for example, promoter, enhancer,
silencer, splice donor or splice acceptor or polyadenylation
signal). Suitable advantageous constructs are described herein
below. Polyadenylation is not required, nor do elements for
initiating translation have to be present.
[0133] A dsRNA can be synthesized chemically or enzymatically.
Cellular RNA polymerases or bacteriophage RNA polymerases (such as,
for example T3, T7 or SP6 RNA polymerase) can be used for this
purpose. Suitable methods for the in-vitro expression of RNA are
described (WO 97/32016; U.S. Pat. No. 5,593,874; U.S. Pat. No.
5,698,425, U.S. Pat. No. 5,712,135, U.S. Pat. No. 5,789,214, U.S.
Pat. No. 5,804,693). Prior to introduction into a cell, tissue or
organism, a dsRNA which has been synthesized in vitro either
chemically or enzymaticcally can be isolated to a higher or lesser
degree from the reaction mixture, for example by extraction,
precipitation, electrophoresis, chromatography or combinations of
these methods. The dsRNA can be introduced directly into the cell
or else be applied extracellularly (for example into the
interstitial space). In one embodiment of the invention the RNAi
method leads to only a partial loss of gene function and therefore
enables the skilled worker to study a gene dose effect in the
desired plant and to fine tune the process of the invention. In
another preferred embodiment it leads to a total loss of function
and therefore increases the production of the fine chemical.
Furthermore it enables a person skilled in the art to study
multiple functions of a gene.
[0134] Stable transformation of the plant with an expression
construct, which brings about the expression of the dsRNA is
preferred, however. Suitable methods are described herein
below.
B) Introduction of an Antisense Nucleic Acid Sequence
[0135] Methods for suppressing a specific protein by preventing the
accumulation of its mRNA by means of "antisense" technology can be
used widely and has been described extensively, including for
plants [Sheehy et al. (1988) Proc. Natl. Acad. Sci. USA 85:
8805-8809; U.S. Pat. No. 4,801,34100; Mol J N et al. (1990) FEBS
Lett 268(2): 427-430]. The antisense nucleic acid molecule
hybridizes with, or binds to, the cellular mRNA and/or the genomic
DNA encoding the target protein to be suppressed. This process
suppresses the transcription and/or translation of the target
protein. Hybridization can be brought about in the conventional
manner via the formation of a stable duplex or, in the case of
genomic DNA, by the antisense nucleic acid molecule binding to the
duplex of the genomic DNA by specific interaction in the large
groove of the DNA helix.
[0136] An "antisense" nucleic acid molecule comprises a nucleotide
sequence, which is at least in part complementary to a "sense"
nucleic acid molecule encoding a protein, e.g., complementary to
the coding strand of a double-stranded cDNA molecule or
complementary to an encoding mRNA sequence. Accordingly, an
antisense nucleic acid molecule can bind via hydrogen bonds to a
sense nucleic acid molecule. The antisense nucleic acid molecule
can be complementary to an entire coding strand of a nucleic acid
molecule conferring the expression of the polypeptide of the
invention or to only a portion thereof. Accordingly, an antisense
nucleic acid molecule can be antisense to a "coding region" of the
coding strand of a nucleotide sequence of a nucleic acid molecule
of the present invention. Advantageously the noncoding region is in
the area of 50 bp, 100 bp, 200 bp or 300 bp, pererably 400 bp, 500
bp, 600 bp, 700 bp, 800 bp, 900 bp or 1000 bp up- and/or downstream
from the coding region. The term "coding region" refers to the
region of the nucleotide sequence comprising codons, which are
translated into amino acid residues. Further, the antisense nucleic
acid molecule is antisense to a "noncoding region" of the mRNA
flanking the coding region of a nucleotide sequence. The term
"noncoding region" refers to 5' and 3' sequences which flank the
coding region that are not translated into a polypeptide, i.e.,
also referred to as 5' and 3' untranslated regions (5'-UTR or
3'-UTR).
[0137] Given the coding strand sequences encoding the polypeptide
of the present invention, e.g. having above mentioned activity,
e.g. the activity of a polypeptide with the biological activity of
the protein of the invention as disclosed herein, antisense nucleic
acid molecules of the invention can be designed according to the
rules of Watson and Crick base pairing.
[0138] An antisense nucleic acid sequence which is suitable for
reducing the activity of a protein can be deduced using the nucleic
acid sequence encoding this protein, for example the nucleic acid
sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ
ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:
15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ
ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ
ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO:
51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ
ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:
69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ
ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:
103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:
111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 (or homologs, analogs, paralogs, orthologs thereof), by
applying the base-pair rules of Watson and Crick. The antisense
nucleic acid sequence can be complementary to all of the
transcribed mRNA of the protein; it may be limited to the coding
region, or it may only consist of one oligonucleotide, which is
complementary to part of the coding or noncoding sequence of the
mRNA. Thus, for example, the oligonucleotide can be complementary
to the nucleic acid region, which encompasses the translation start
for the protein. Antisense nucleic acid sequences may have an
advantageous length of, for example, 5, 10, 15, 20, 25, 30, 35, 40,
45 or 50 nucleotides but they may also be longer and encompass at
least 100, 200, 500, 1000, 2000 or 5000 nucleotides. A particular
preferred length is between 15 and 30 nucleotides such as 15, 20,
25 or 30 nucleotides. Antisense nucleic acid sequences can be
expressed recombinantly or synthesized chemically or enzymatically
using methods known to the skilled worker. For example, an
antisense nucleic acid molecule (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides can be used. Examples of
substances which can be used are phosphorothioate derivatives and
acridine-substituted nucleotides such as 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthin, xanthin,
4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil,
5-carboxymethyl-aminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
.beta.-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
.beta.-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid,
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid molecule has been subcloned in an antisense
orientation (i.e., RNA transcribed from the inserted nucleic acid
molecule will be of an antisense orientation to a target nucleic
acid molecule of interest, described further in the following
subsection).
[0139] In a further preferred embodiment, the expression of a
protein encoded by one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 or homologs, analogs, paralogs, orthologs thereof can be
inhibited by nucleotide sequences which are complementary to the
regulatory region of a gene (for example a promoter and/or
enhancer) and which may form triplex structures with the DNA double
helix in this region so that the transcription of the gene is
reduced. Such methods have been described (Helene C (1991)
Anticancer Drug Res. 6(6): 569-84; Helene C et al. (1992) Ann. NY
Acad. Sci. 660: 27-36; Maher L J (1992) Bioassays 14(12):
807-815).
[0140] In a further embodiment, the antisense nucleic acid molecule
can be an .alpha.-anomeric nucleic acid. Such a-anomeric nucleic
acid molecules form specific double-stranded hybrids with
complementary RNA in which--as opposed to the conventional
.beta.-nucleic acids--the two strands run in parallel with one
another (Gautier C et al. (1987) Nucleic Acids Res. 15: 6625-6641).
Furthermore, the antisense nucleic acid molecule can also comprise
2'-O-methylribonucleotides [Inoue et al. (1987) Nucleic Acids Res.
15: 6131-6148] or chimeric RNA-DNA analogs [Inoue et al. (1987)
FEBS Lett 215: 327-330].
[0141] The antisense nucleic acid molecules of the invention are
typically administered to a cell or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a polypeptide having the biological activity of protein of
the invention thereby inhibit expression of the protein, e.g., by
inhibiting transcription and/or translation and leading to the
aforementioned yield increasing activity.
[0142] The antisense molecule of the present invention comprises
also a nucleic acid molecule comprising a nucleotide sequences
complementary to the regulatory region of an nucleotide sequence
encoding the natural occurring polypeptide of the invention, e.g.
the polypeptide sequences shown in the sequence listing, or
identified according to the methods described herein, e.g., its
promoter and/or enhancers, e.g. to form triple helical structures
that prevent transcription of the gene in target cells. See
generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;
Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher,
L. J. (1992) Bioassays 14(12):807-15.
C) Introduction of an Antisense Nucleic Acid Sequence Combined with
a Ribozyme
[0143] It is advantageous to combine the above-described antisense
strategy with a ribozyme method. Catalytic RNA molecules or
ribozymes can be adapted to any target RNA and cleave the
phosphodiester backbone at specific positions, thus functionally
deactivating the target RNA (Tanner N K (1999) FEMS Microbiol. Rev.
23(3): 257-275). The ribozyme per se is not modified thereby, but
is capable of cleaving further target RNA molecules in an analogous
manner, thus acquiring the properties of an enzyme. The
incorporation of ribozyme sequences into "antisense" RNAs imparts
this enzyme-like RNA-cleaving property to precisely these
"antisense" RNAs and thus increases their efficiency when
inactivating the target RNA. The preparation and the use of
suitable ribozyme "antisense" RNA molecules is described, for
example, by Haseloff et al. (1988) Nature 33410: 585-591.
[0144] Further the antisense nucleic acid molecule of the invention
can be also a ribozyme. Ribozymes are catalytic RNA molecules with
ribonuclease activity, which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. In this manner, ribozymes [for example
"Hammerhead" ribozymes; Haselhoff and Gerlach (1988) Nature 33410:
585-591] can be used to catalytically cleave the mRNA of an enzyme
to be suppressed and to prevent translation. The ribozyme
technology can increase the efficacy of an antisense strategy.
Methods for expressing ribozymes for reducing specific proteins are
described in (EP 0 291 533, EP 0 321 201, EP 0 360 257). Ribozyme
expression has also been described for plant cells (Steinecke P et
al. (1992) EMBO J 11(4): 1525-1530; de Feyter R et al. (1996) Mol.
Gen. Genet. 250(3): 329-338). Suitable target sequences and
ribozymes can be identified for example as described by Steinecke
P, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds,
Academic Press, Inc. (1995), pp. 449-460 by calculating the
secondary structures of ribozyme RNA and target RNA and by their
interaction [Bayley C C et al. (1992) Plant Mol. Biol. 18(2):
353-361; Lloyd AM and Davis R W et al. (1994) Mol. Gen. Genet.
242(6): 653-657]. For example, derivatives of the tetrahymena L-19
IVS RNA, which have complementary regions to the mRNA of the
protein to be suppressed can be constructed (see also U.S. Pat. No.
4,987,071 and U.S. Pat. No. 5,116,742). As an alternative, such
ribozymes can also be identified from a library of a variety of
ribozymes via a selection process (Bartel D and Szostak J W (1993)
Science 261: 1411-1418).
D) Introduction of a (Sense) Nucleic Acid Sequence for Inducing
Cosuppression
[0145] The expression of a nucleic acid sequence in sense
orientation can lead to cosuppression of the corresponding
homologous, endogenous genes. The expression of sense RNA with
homology to an endogenous gene can reduce or indeed eliminate the
expression of the endogenous gene, in a similar manner as has been
described for the following antisense approaches: Jorgensen et al.
[(1996) Plant Mol. Biol. 31(5): 957-973], Goring et al. [(1991)
Proc. Natl. Acad. Sci. USA 88: 1770-1774], Smith et al. [(1990)
Mol. Gen. Genet. 224: 447-481], Napoli et al. [(1990) Plant Cell 2:
279-289] or Van der Krol et al. [(1990) Plant Cell 2: 291-99]. In
this context, the construct introduced may represent the homologous
gene to be reduced either in full or only in part. The application
of this technique to plants has been described for example by
Napoli et al. [(1990) The Plant Cell 2: 279-289 and in U.S. Pat.
No. 5,034,323]. Furthermore the above described cosuppression
strategy can advantageously be combined with the RNAi method as
described by Brummell et al., 2003, Plant J. 33, pp 793-800.
E) Introduction of Nucleic Acid Sequences Encoding a
Dominant-Negative Protein
[0146] The function or activity of a protein can efficiently also
be reduced by expressing a dominant-negative variant of said
protein. The skilled worker is familiar with methods for reducing
the function or activity of a protein by means of coexpression of
its dominant-negative form [Lagna G and Hemmati-Brivanlou A (1998)
Current Topics in Developmental Biology 36: 75-98; Perlmutter R M
and Alberola-Ila J (1996) Current Opinion in Immunology 8(2):
285-90; Sheppard D (1994) American Journal of Respiratory Cell
& Molecular Biology 11(1): 1-6; Herskowitz I (1987) Nature 329
(6136): 219-22].
[0147] A dominant-negative variant can be realized for example by
changing of an amino acid in the proteins encoded by one of SEQ ID
NO: 2 or homologs thereof. This change can be determined for
example by computer-aided comparison ("alignment"). These mutations
for achieving a dominant-negative variant are preferably carried
out at the level of the nucleic acid sequences. A corresponding
mutation can be performed for example by PCR-mediated in-vitro
mutagenesis using suitable oligonucleotide primers by means of
which the desired mutation is introduced. To this end, methods are
used with which the skilled worker is familiar. For example, the
"LA PCR in vitro Mutagenesis Kit" (Takara Shuzo, Kyoto) can be used
for this purpose. It is also possible and known to those skilled in
the art that deleting or changing of functional domains, e.g. TF or
other signaling components which can bind but not activate may
achieve the reduction of protein activity.
F) Introduction of DNA- or Protein-Binding Factors Against Genes,
RNAs or Proteins
[0148] A reduction in the expression of a gene encoded by one of
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ
ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:
27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ
ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ
ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:
63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ
ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:
81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105,
SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ
ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID
NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:
187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:
195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:
203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:
211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:
219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:
227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:
235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:
243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO:
251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO:
275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO:
283, SEQ ID NO: 285 or SEQ ID NO: 287 or homologs thereof according
to the invention can also be achieved with specific DNA-binding
factors, for example factors of the zinc finger transcription
factor type. These factors attach to the genomic sequence of the
endogenous target gene, preferably in the regulatory regions, and
bring about repression of the endogenous gene. The use of such a
method makes possible the reduction in the expression of an
endogenous gene without it being necessary to recombinantly
manipulate the sequence of the latter. Such methods for the
preparation of relevant factors are described in Dreier B et al.
[(2001) J. Biol. Chem. 276(31): 29466-78 and (2000) J. Mol. Biol.
303(4): 489-502], Beerli R R et al. [(1998) Proc. Natl. Acad. Sci.
USA 95(25): 14628-14633; (2000) Proc. Natl. Acad. Sci. USA 97(4):
1495-1500 and (2000) J. Biol. Chem. 275(42): 32617-32627)], Segal D
J and Barbas C F [3rd (2000) Curr. Opin. Chem. Biol. 4(1):
3410-39], Kang J S and Kim J S [(2000) J. Biol. Chem. 275(12):
8742-8748], Kim J S et al. [(1997) Proc. Natl. Acad. Sci. USA
94(8): 3616-3620], Klug A [(1999) J. Mol. Biol. 293(2): 215-218],
Tsai S Y et al. [(1998) Adv. Drug Deliv. Rev. 30(1-3): 23-31], Mapp
A K et al. [(2000) Proc. Natl. Acad. Sci. USA 97(8): 3930-3935],
Sharrocks A D et al. [(1997) Int. J. Biochem. Cell Biol. 29(12):
1371-1387] and Zhang L et al. [(2000) J. Biol. Chem. 275(43):
33850-33860]. Examples for the application of this technology in
plants have been described in WO 01/52620, Ordiz M I et al., (Proc.
Natl. Acad. Sci. USA, Vol. 99, Issue 20, 13290-13295, 2002) or Guan
et al., (Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 20,
13296-13301, 2002)
[0149] These factors can be selected using any portion of a gene.
This segment is preferably located in the promoter region. For the
purposes of gene suppression, however, it may also be located in
the region of the coding exons or introns. The skilled worker can
obtain the relevant segments from Genbank by database search or
starting from a cDNA whose gene is not present in Genbank by
screening a genomic library for corresponding genomic clones.
[0150] It is also possible to first identify sequences in a target
crop, which are encoded by one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ
ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID
NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID
NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID
NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ
ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID
NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 or homologs thereof, then find the promoter and reduce
expression by the use of the abovementioned factors.
[0151] The skilled worker is familiar with the methods required for
doing so.
[0152] Furthermore, factors which are introduced into a cell may
also be those which themselves inhibit the target protein. The
protein-binding factors can, for example, be aptamers [Famulok M
and Mayer G (1999) Curr. Top Microbiol. Immunol. 243: 123-36] or
antibodies or antibody fragments or single-chain antibodies.
Obtaining these factors has been described, and the skilled worker
is familiar therewith. For example, a cytoplasmic scFv anti-body
has been employed for modulating activity of the phytochrome A
protein in genetically modified tobacco plants [Owen M et al.
(1992) Biotechnology (NY) 10(7): 790-794; Franken E et al. (1997)
Curr. Opin. Biotechnol. 8(4): 411-416; Whitelam (1996) Trend Plant
Sci. 1: 286-272].
[0153] Gene expression may also be suppressed by tailor-made
low-molecular-weight synthetic compounds, for example of the
polyamide type [Dervan P B and Burli R W (1999) Current Opinion in
Chemical Biology 3: 688-693; Gottesfeld J M et al. (2000) Gene
Expr. 9(1-2): 77-91]. These oligomers consist of the units
3-(dimethyl amino)propylamine, N-methyl-3-hydroxypyrrole,
N-methylimidazole and N-methylpyrroles; they can be adapted to each
portion of double-stranded DNA in such a way that they bind
sequence-specifically to the large groove and block the expression
of the gene sequences located in this position. Suitable methods
have been described in Bremer R E et al. [(2001) Bioorg. Med. Chem.
9(8): 2093-103], Ansari A Z et al. [(2001) Chem. Biol. 8(6):
583-92], Gottesfeld J M et al. [(2001) J. Mol. Biol. 309(3):
615-29], Wurtz N R et al. [(2001) Org. Lett 3(8): 1201-3], Wang C C
et al. [(2001) Bioorg. Med. Chem. 9(3): 653-7], Urbach A R and
Dervan P B [(2001) Proc. Natl. Acad. Sci. USA 98(8): 434103-8] and
Chiang S Y et al. [(2000) J. Biol. Chem. 275(32): 24246-54].
G) Introduction of Viral Nucleic Acid Sequences and Expression
Constructs which Bring about the Degradation of RNA
[0154] Inactivation or downregulation can also be efficiently
brought about by inducing specific RNA degradation by the organism,
advantageously in the plant, with the aid of a viral expression
system (Amplikon) [Angell, S M et al. (1999) Plant J. 20(3):
357-362]. Nucleic acid sequences with homology to the transcripts
to be suppressed are introduced into the plant by these
systems--also referred to as "VIGS" (viral induced gene silencing)
with the aid of viral vectors. Then, transcription is switched off,
presumably mediated by plant defense mechanisms against viruses.
Suitable techniques and methods are described in Ratcliff F et al.
[(2001) Plant J. 25(2): 237-45], Fagard M and Vaucheret H [(2000)
Plant Mol. Biol. 43(2-3): 285-93], Anandalakshmi R et al. [(1998)
Proc. Natl. Acad. Sci. USA 95(22): 13079-84] and Ruiz M T [(1998)
Plant Cell 10(6): 937-46].
H) Introduction of Constructs for Inducing a Homologous
Recombination on Endogenous Genes, for Example for Generating
Knock-Out Mutants
[0155] To generate a homologously-recombinant organism with reduced
activity, a nucleic acid construct is used which, for example,
comprises at least part of an endogenous gene which is modified by
a deletion, addition or substitution of at least one nucleotide in
such a way that the functionality is reduced or completely
eliminated. The modification may also affect the regulatory
elements (for example the promoter) of the gene so that the coding
sequence remains unmodified, but expression (transcription and/or
translation) does not take place or is reduced.
[0156] In the case of conventional homologous recombination, the
modified region is flanked at its 5' and 3' end by further nucleic
acid sequences, which must be sufficiently long for allowing
recombination. Their length is, as a rule, in a range of from one
hundred bases up to several kilobases [Thomas K R and Capecchi M R
(1987) Cell 51: 503; Strepp et al. (1998) Proc. Natl. Acad. Sci.
USA 95(8): 4368-4373]. In the case of homologous recombination, the
host organism--for example a plant--is transformed with the
recombination construct using the methods described herein below,
and clones, which have successfully undergone recombination are
selected using for example a resistance to antibiotics or
herbicides. Using the cotransformation technique, the resistance to
antibiotics or herbicides can subsequently advantageously be
re-eliminated by performing crosses. An example for an efficient
homologous recombination system in plants has been published in
Nat. Biotechnol. 2002 October; 20(10):1030-4, Terada R et al.:
Efficient gene targeting by homologous recombination in rice.
[0157] Homologous recombination is a relatively rare event in
higher eukaryotes, especially in plants. Random integrations into
the host genome predominate. One possibility of removing the
randomly integrated sequences and thus increasing the number of
cell clones with a correct homologous recombination is the use of a
sequence-specific recombination system as described in U.S. Pat.
No. 6,110,736, by means of which unspecifically integrated
sequences can be deleted again, which simplifies the selection of
events which have integrated successfully via homologous
recombination. A multiplicity of sequence-specific recombination
systems may be used, examples which may be mentioned being Cre/lox
system of bacteriophage P1, the FLP/FRT system from yeast, the Gin
recombinase of phage Mu, the Pin recombinase from E. coli and the
R/RS system of the pSR1 plasmid. The bacteriophage P1 Cre/lox
system and the yeast FLP/FRT system are preferred. The FLP/FRT and
the cre/lox recombinase system have already been applied to plant
systems [Odell et al. (1990) Mol. Gen. Genet. 223: 369-378].
I) Introduction of Mutations into Endogenous Genes for Bringing
about a Loss of Function (for Example Generation of Stop Codons,
Reading-Frame Shifts and the Like)
[0158] Further suitable methods for reducing activity are the
introduction of non-sense mutations into endogenous genes, for
example by introducing RNA/DNA oligonucleotides into the plant [Zhu
et al. (2000) Nat. Biotechnol. 18(5): 555-558], and the generation
of knock-out mutants with the aid of, for example, T-DNA
mutagenesis [Koncz et al. (1992) Plant Mol. Biol. 20(5): 963-976],
ENU-(N-ethyl-N-nitrosourea)--mutagenesis or homologous
recombination [Hohn B and Puchta (1999) H. Proc. Natl. Acad. Sci.
USA 96: 8321-8323]. Point mutations may also be generated by means
of DNA-RNA hybrids also known as "chimeraplasty" [Cole-Strauss et
al. (1999) Nucl. Acids Res. 27(5): 1323-1330; Kmiec (1999) Gene
Therapy American Scientist 87(3): 240-247]. The mutation sites may
be specifically targeted or randomly selected. If the mutations
have been created randomly e.g. by Transposon-Tagging or chemical
mutagenesis, the skilled worked is able to specifically enrich
selected muation events in the inventive nucleic acids.
[0159] Nucleic acid sequences as described in item B) to I) are
expressed in the cell or organism by transformation/transfection of
the cell or organism or are introduced in the cell or organism by
known methods, for example as disclosed in item A).
[0160] In one further embodiment of the process according to the
invention, organisms are used in which one of the abovementioned
genes, or one of the abovementioned nucleic acids, is mutated in
such a manner that the activity of the encoded gene products is
influenced by cellular factors to a greater extent than in the
reference organism, as compared with the unmutated proteins. This
kind of mutation could lead to an endogenous down-regulation of
activity of the polypeptides of the invention which than causes an
yield increase. In a further embodiment the process according to
the invention, organisms are grown under such conditions, that the
expression of the nucleic acids of the invention is reduced or
repressed leading to an yield increase according to the
invention.
[0161] Accordingly, in one embodiment, the process according to the
invention relates to a process which comprises [0162] a) providing
a plant cell, a plant tissue or a plant; [0163] b) reducing,
decreasing or deleting the activity in the plant cell, the plant
tissue or the plant, of a protein having the biological activity of
the protein of the invention or being encoded by the nucleic acid
molecule of the present invention and described below, e.g. having
above mentioned activity, e.g. conferring an increase in yield;
[0164] c) growing the plant cell, the plant tissue or the plant
under conditions which permit the increase in yield of the plant
cell, the plant tissue or the plant; and [0165] d) if desired,
recovering the plant cell, the plant tissue, the fruit, the seed,
the root, the tubers, the leaves, the blossoms or the whole
plant.
[0166] After the above-described reducing, decreasing or deleting
(which as defined above also encompasses the generating of an
activity in an organism, i.e. a de novo activity), for example
after the introduction and the expression of the an RNAi molecule,
antisense molecule or ribozyme described in the methods or
processes according to the invention, the organism according to the
invention, advantageously, a plant, plant tissue or plant cell, is
grown and subsequently harvested.
[0167] In the event that the transgenic host organism is a plant,
plant tissue or plant cell such as plants selected from the group
consisting of the families Anacardiaceae, Asteraceae, Apiaceae,
Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae,
Cannabaceae, Convolvulaceae, Chenopodiaceae, Cucurbitaceae,
Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae,
Gramineae, Juglandaceae, Lauraceae, Leguminosae, Linaceae or
perennial grass, fodder crops, vegetables, ornamentals and
Arabidopsis thaliana. Such plants are either grown on a solid
medium or as cells in a liquid medium, which is known to the
skilled worker and suits the organism. Furthermore such plants can
be grown in soil or the like. After the growing phase, the plants
can be harvested. The plants or plant cells or the recovered, and
if desired isolated plant parts as fruits, seeds, roots, tubers,
leaves, blossoms can then be processed further directly into
foodstuffs or animal feeds or for other applications.
[0168] Anacardiaceae such as the genera Pistacia, Mangifera,
Anacardium e.g. the species Pistacia vera [pistachios, Pistazie],
Mangifer indica [Mango] or Anacardium occidentale [Cashew];
Asteraceae such as the genera Calendula, Carthamus, Centaurea,
Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana
e.g. the species Calendula officinalis [Marigold], Carthamus
tinctorius [safflower], Centaurea cyanus [cornflower], Cichorium
intybus [blue daisy], Cynara scolymus [Artichoke], Helianthus annus
[sunflower], Lactuca sativa, Lactuca crispa, Lactuca esculenta,
Lactuca scariola L. ssp. sativa, Lactuca scariola L. var.
integrata, Lactuca scariola L. var. integrifolia, Lactuca sativa
subsp. romana, Locusta communis, Valeriana locusta [lettuce],
Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [Marigold];
Apiaceae such as the genera Daucus e.g. the species Daucus carota
[carrot]; Betulaceae such as the genera Corylus e.g. the species
Corylus avellana or Corylus colurna [hazelnut]; Boraginaceae such
as the genera Borago e.g. the species Borago officinalis [borage];
Brassicaceae such as the genera Brassica, Melanosinapis, Sinapis,
Arabidopsis e.g. the species Brassica napus, Brassica rapa ssp.
[canola, oilseed rape, turnip rape], Sinapis arvensis Brassica
juncea, Brassica juncea var. juncea, Brassica juncea var.
crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassica
sinapioides, Melanosinapis communis [mustard], Brassica oleracea
[fodder beet] or Arabidopsis thaliana; Bromeliaceae such as the
genera Anana, Bromelia e.g. the species Anana comosus, Ananas
ananas or Bromelia comosa [pineapple]; Caricaceae such as the
genera Carica e.g. the species Carica papaya [papaya]; Cannabaceae
such as the genera Cannabis e.g. the species Cannabis sative
[hemp], Convolvulaceae such as the genera Ipomea, Convolvulus e.g.
the species Ipomoea batatus, Ipomoea pandurata, Convolvulus
batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea
tiliacea, Ipomoea triloba or Convolvulus panduratus [sweet potato,
Man of the Earth, wild potato], Chenopodiaceae such as the genera
Beta, i.e. the species Beta vulgaris, Beta vulgaris var. altissima,
Beta vulgaris var. Vulgaris, Beta maritima, Beta vulgaris var.
perennis, Beta vulgaris var. conditiva or Beta vulgaris var.
esculenta [sugar beet]; Cucurbitaceae such as the genera Cucubita
e.g. the species Cucurbita maxima, Cucurbita mixta, Cucurbita pepo
or Cucurbita moschata [pumpkin, squash]; Elaeagnaceae such as the
genera Elaeagnus e.g. the species Olea europaea [olive]; Ericaceae
such as the genera Kalmia e.g. the species Kalmia latifolia, Kalmia
angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmia
occidentalis, Cistus chamaerhodendros or Kalmia lucida [American
laurel, broad-leafed laurel, calico bush, spoon wood, sheep laurel,
alpine laurel, bog laurel, western bog-laurel, swamp-laurel];
Euphorbiaceae such as the genera Manihot, Janipha, Jatropha,
Ricinus e.g. the species Manihot utilissima, Janipha manihot,
Jatropha manihot, Manihot aipil, Manihot dulcis, Manihot manihot,
Manihot melanobasis, Manihot esculenta [manihot, arrowroot,
tapioca, cassava] or Ricinus communis [castor bean, Castor Oil
Bush, Castor Oil Plant, Palma Christi, Wonder Tree]; Fabaceae such
as the genera Pisum, Albizia, Cathormion, Feuillea, Inga,
Pithecolobium, Acacia, Mimosa, Medicajo, Glycine, Dolichos,
Phaseolus, Soja e.g. the species Pisum sativum, Pisum arvense,
Pisum humile [pea], Albizia berteriana, Albizia julibrissin,
Albizia lebbeck, Acacia berteriana, Acacia littoralis, Albizia
berteriana, Albizzia berteriana, Cathormion berteriana, Feuillea
berteriana, Ingafragrans, Pithecellobiwn berterianum,
Pithecellobiumfragrans, Pithecolobium berterianum, Pseudalbizzia
berteriana, Acacia julibrissin, Acacia nemu, Albizia nemu,
Feuilleea julibrissin, Mimosa julibrissin, Mimosa speciosa,
Sericanrda julibrissin, Acacia lebbeck, Acacia macrophylla, Albizia
lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa [bastard
logwood, silk tree, East Indian Walnut], Medicago sativa, Medicago
falcata, Medicago varia [alfalfa] Glycine max Dolichos soja,
Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida or
Soja max [soybean]; Geraniaceae such as the genera Pelargonium,
Cocos, Oleum e.g. the species Cocos nucifera, Pelargonium
grossularioides or Oleum cocois [coconut]; Gramineae such as the
genera Saccharum e.g. the species Saccharum officinarum;
Juglandaceae such as the genera Juglans, Wallia e.g. the species
Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans
cinerea, Wallia cinerea, Juglans bixbyi, Juglans califormica,
Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans
major, Juglans microcarpa, Juglans nigra or Wallia nigra [walnut,
black walnut, common walnut, persian walnut, white walnut,
butternut, black walnut]; Lauraceae such as the genera Persea,
Laurus e.g. the species laurel Laurus nobilis [bay, laurel, bay
laurel, sweet bay], Persea americana Persea americana, Persea
gratissima or Persea persea [avocado]; Leguminosae such as the
genera Arachis e.g. the species Arachis hypogaea [peanut]; Linaceae
such as the genera Linum, Adenolinum e.g. the species Linum
usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum
angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum,
Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum
perenne, Linum perenne var. lewisii, Linum pratense or Linum
trigynum [flax, linseed]; Lythrarieae such as the genera Punica
e.g. the species Punica granatum [pomegranate]; Malvaceae such as
the genera Gossypium e.g. the species Gossypium hirsutum, Gossypium
arboreum, Gossypium barbadense, Gossypium herbaceum or Gossypium
thurberi [cotton]; Musaceae such as the genera Musa e.g. the
species Musa nana, Musa acuminata, Musa paradisiaca, Musa spp.
[banana]; Onagraceae such as the genera Camissonia, Oenothera e.g.
the species Oenothera biennis or Camissonia brevipes [primrose,
evening primrose]; Palmae such as the genera Elacis e.g. the
species Elaeis guineensis [oil plam]; Papaveraceae such as the
genera Papaver e.g. the species Papaver orientale, Papaver rhoeas,
Papaver dubium [poppy, oriental poppy, corn poppy, field poppy,
shirley poppies, field poppy, long-headed poppy, long-pod poppy];
Pedaliaceae such as the genera Sesamum e.g. the species Sesamum
indicum [sesame]; Piperaceae such as the genera Piper, Artanthe,
Peperomia, Steffensia e.g. the species Piper aduncum, Piper
amalago, Piper angustifolium, Piper auritum, Piper betel, Piper
cubeba, Piper longum, Piper nigrum, Piper retrofractum, Artanthe
adunca, Artanthe elongata, Peperomia elongata, Piper elongatum,
Steffensia elongata. [Cayenne pepper, wild pepper]; Poaceae such as
the genera Hordeum, Secale, Avena, Sorghum, Andropogon, Holcus,
Panicum, Oryza, Zea, Triticum e.g. the species Hordeum vulgare,
Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum
distichon Hordeum aegiceras, Hordeum hexastichon, Hordeum
hexastichum, Hordeum irregulare, Hordeum sativum, Hordeum secalinum
[barley, pearl barley, foxtail barley, wall barley, meadow barley],
Secale cereale [rye], Avena sativa, Avena fatua, Avena byzantina,
Avena fatua var. sativa, Avena hybrida [oat], Sorghum bicolor,
Sorghum halepense, Sorghum saccharatum, Sorghum vulgare, Andropogon
drummondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum,
Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum, Sorghum
dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,
Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum
subglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcus
halepensis, Sorghum miliaceum millet, Panicum militaceum [Sorghum,
millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn,
maize] Triticum aestivum, Triticum durum, Triticum turgidum,
Triticum hybernum, Triticum macha, Triticum sativum or Triticum
vulgare [wheat, bread wheat, common wheat], Proteaceae such as the
genera Macadamia e.g. the species Macadamia intergrifolia
[macadamia]; Rubiaceae such as the genera Coffea e.g. the species
Cofea spp., Coffea arabica, Coffea canephora or Coffea liberica
[coffee]; Scrophulariaceae such as the genera Verbascum e.g. the
species Verbascum blattaria, Verbascum chaixii, Verbascum
densiflorum, Verbascum lagurus, Verbascum longifolium, Verbascum
lychnitis, Verbascum nigrum, Verbascum olympicum, Verbascum
phlomoides, Verbascum phoenicum, Verbascum pulverulentum or
Verbascum thapsus [mullein, white moth mullein, nettle-leaved
mullein, dense-flowered mullein, silver mullein, long-leaved
mullein, white mullein, dark mullein, greek mullein, orange
mullein, purple mullein, hoary mullein, great mullein]; Solanaceae
such as the genera Capsicum, Nicotiana, Solanum, Lycopersicon e.g.
the species Capsicum annuum, Capsicum annuum var. glabriusculum,
Capsicum frutescens [pepper], Capsicum annuum [paprika], Nicotiana
tabacum, Nicotiana alata, Nicotiana attenuata, Nicotiana glauca,
Nicotiana langsdorffii, Nicotiana obtusifolia, Nicotiana
quadrivalvis, Nicotiana repanda, Nicotiana rustica, Nicotiana
sylvestris [tobacco], Solanum tuberosum [potato], Solanum melongena
[egg-plant] (Lycopersicon esculentum, Lycopersicon lycopersicum,
Lycopersicon pyriforme, Solanum integrifolium or Solanum
lycopersicum [tomato]; Sterculiaceae such as the genera Theobroma
e.g. the species Theobroma cacao [cacao]; Theaceae such as the
genera Camellia e.g. the species Camellia sinensis) [tea]. All
abovementioned host organisms are also useable as source organisms
for the nucleic acid sequences of the invention.
[0169] Particular preferred plants are plants selected from the
group consisting of maize, soja, canola, wheat, barley, triticale,
rice, linseed, sunflower, potato and Arabidopsis.
[0170] With regard to the nucleic acid sequences as depicted in SEQ
ID NO: 1 or SEQ ID NO: 113 a nucleic acid construct which contains
one of said nucleic acid sequences or an organism (=transgenic
organism) which is transformed with one of said nucleic acid
sequences or one of said nucleic acid constructs, "transgene" means
all those constructs which have been brought about by genetic
manipulation methods and in which either [0171] a) the nucleic acid
sequences as depicted in SEQ ID NO: 1, SEQ ID NO: 113 or a
derivative thereof, or [0172] b) a genetic regulatory element, for
example a promoter, which is functionally linked to one of said
nucleic acid sequences as depicted in SEQ ID NO: 1, SEQ ID NO: 113
or a derivative thereof, or [0173] c) (a) and (b)
[0174] is/are not present in its/their natural genetic environment
or has/have been modified by means of genetic manipulation methods,
it being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide radicals. "Natural genetic environment" means the
natural chromosomal locus in the organism of origin or the presence
in a genomic library. In the case of a genomic library, the
natural, genetic environment of the nucleic acid sequence is
preferably at least partially still preserved. The environment
flanks the nucleic acid sequence at least on one side and has a
sequence length of at least 50 bp, preferably at least 500 bp,
particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[0175] However, transgenic also means that the nucleic acids
according to the invention are located at their natural position in
the genome of an organism, but that the sequence has been modified
in comparison with the natural sequence and/or that the regulatory
sequences of the natural sequences have been modified. Preferably,
transgenic/recombinant is to be understood as meaning the
expression of the nucleic acids used in the process according to
the invention in a non-natural position in the genome, that is to
say the expression of the nucleic acids is homologous or,
preferably, heterologous. This expression can be transiently or of
a sequence integrated stably into the genome.
[0176] The use of the nucleic acid sequence according to the
invention or of the nucleic acid construct according to the
invention for the generation of transgenic plants is therefore also
subject matter of the invention.
[0177] Another embodiment of the invention is a process for the
modification of the nucleic acid molecules of the invention encoded
by the host organism for example by random mutagenesis with
chemicals, radiation or UV-light or side directed mutagenesis in
such a manner that the yield of the plant is increased. This
embodiment of the invention shall be deemed as transgenic in the
sense of the invention.
[0178] In this context, the yield of the plant or plant part of the
invention may be increased according to the process of the
invention by at least a factor of 1.1, preferably at least a factor
of 1.2; 1.3; 1.4; or 1.5, especially preferably by at least a
factor of 1.6 or 17.5, very especially preferably by at least a
factor of 2, in comparison with the wild type, control or
reference. Preferably, said increase is found in a tissue, more
preferred in a plant or in a harvestable part thereof
[0179] In the inventive process as mentioned above preferably the
reduction, decrease or deletion of the biological activity
represented by protein of the invention is achieved by reducing,
decreasing or deleting the expression of at least one nucleic acid
molecule, wherein the nucleic acid molecule is selected from the
group consisting of: [0180] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide shown in
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:
28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ
ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ
ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:
64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ
ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:
82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106,
SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ
ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID
NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288; [0181] b) nucleic acid
molecule comprising, preferably at least the mature form, of the
nucleic acid molecule shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID
NO: 23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID
NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID
NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ
ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID
NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287; [0182] c) nucleic acid molecule comprising a nucleic acid
sequence, which, as a result of the degeneracy of the genetic code,
can be derived from a polypeptide sequence depicted in SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,
SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID
NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,
SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID
NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,
SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID
NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,
SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID
NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO:
108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288 and having the biological
activity represented by protein as depicted in SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID
NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,
SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID
NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,
SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID
NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84,
SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ
ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID
NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288; [0183] d) nucleic acid molecule encoding a
polypeptide having at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and having the biological activity represented by
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; [0184] e) nucleic acid molecule which comprises a
polynucleotide which is obtained by amplifying a cDNA library or a
genomic library using the primers depicted in SEQ ID NO: 92 and SEQ
ID NO: 93 and having the biological activity represented by protein
as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:
44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ
ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:
62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ
ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:
80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104,
SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 or
using the primers depicted in 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 or SEQ ID NO: 264 and having the biological activity of SEQ ID
NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288; [0185] f)
nucleic acid molecule which encodes a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (b)
or (c) and encoding a polypeptide having the biological activity
represented by protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288;
[0186] g) nucleic acid molecule which encodes a fragment or an
epitope of a polypeptide which is encoded by one of the nucleic
acid molecules of (a) to (e), preferably to (a) to (b) or (c) and
encoding a polypeptide having the biological activity represented
by protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288; [0187] h) nucleic acid molecule encoding a polypeptide which
is isolated with the aid of monoclonal or polyclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (e), preferably to (a) to (b) or (c), and having the
biological activity represented by the protein as depicted in SEQ
ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID
NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28,
SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID
NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46,
SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID
NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64,
SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID
NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,
SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ
ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID
NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288; [0188] i) nucleic acid
molecule encoding a polypeptide comprising the consensus sequence
shown in shown in SEQ ID NO: 87 or SEQ ID NO: 88 or SEQ ID NO: 89
or SEQ ID NO: 90 or SEQ ID NO: 91 or SEQ ID NO: 265 or SEQ ID NO:
266 or SEQ ID NO: 267 or SEQ ID NO: 268 and having the biological
activity represented by the protein as depicted in SEQ ID NO: 2 or
SEQ ID NO: 113; [0189] j) nucleic acid molecule encoding a
polypeptide having the biological activity represented by the
protein SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ
ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:
18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ
ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:
36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ
ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:
54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ
ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO:
72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ
ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO:
106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288; and [0190]
k) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridisation
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) or (b) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(c) and encoding a polypeptide having the biological activity
represented by protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288;
[0191] or which comprises a sequence which is complementary
thereto.
[0192] In one embodiment, the nucleic acid molecule used in the
process distinguishes over the sequence depicted in SEQ ID NO: 1 or
SEQ ID NO: 113 by at least one or more nucleotides or does not
consist of the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 113. In
one embodiment, the nucleic acid molecule of the present invention
is less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence shown in SEQ ID NO: 1 or SEQ ID NO: 113. In another
embodiment, the nucleic acid molecule does not consist of the
sequence shown in SEQ ID NO: 1 or SEQ ID NO: 113.
[0193] Unless otherwise specified, the terms "polynucleotides",
"nucleic acid" and "nucleic acid molecule" are interchangeably in
the present context. Unless otherwise specified, the terms
"peptide", "polypeptide" and "protein" are interchangeably in the
present context. The term "sequence" may relate to polynucleotides,
nucleic acids, nucleic acid molecules, peptides, polypeptides and
proteins, depending on the context in which the term "sequence" is
used. The terms "gene(s)", "polynucleotide", "nucleic acid
sequence", "nucleotide sequence", or "nucleic acid molecule(s)" as
used herein refers to a polymeric form of nucleotides of any
length, either ribonucleotides or deoxyribonucleotides. The terms
refer only to the primary structure of the molecule.
[0194] Thus, the terms "gene(s)", "polynucleotide", "nucleic acid
sequence", "nucleotide sequence", or "nucleic acid molecule(s)" as
used herein include double- and single-stranded DNA and RNA. They
also include known types of modifications, for example,
methylation, "caps", substitutions of one or more of the naturally
occurring nucleotides with an analog. Preferably, the DNA or RNA
sequence of the invention comprises a coding sequence encoding the
herein defined polypeptide.
[0195] A "coding sequence" is a nucleotide sequence, which is
transcribed into mRNA and/or translated into a polypeptide when
placed under the control of appropriate regulatory sequences. The
boundaries of the coding sequence are determined by a translation
start codon at the 5'-terminus and a translation stop codon at the
3'-terminus. A coding sequence can include, but is not limited to
mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while
introns may be present as well under certain circumstances.
[0196] Nucleic acid molecules with the sequence shown in SEQ ID NO:
1 or SEQ ID NO: 113, nucleic acid molecules which are derived from
the amino acid sequences shown in SEQ ID NO: 2 or SEQ ID NO: 114 or
their derivatives or homologues encoding polypeptides with the
enzymatic or biological activity of protein of the invention or
conferring the yield increase after reducing, decreasing or
deleting its expression or activity in the process according to the
invention. These sequences are cloned in such a manner into nucleic
acid constructs that their activity is reduced, decreased or
deleted, either individually or in combination with other sequences
involved in yield. These nucleic acid constructs enable an optimal
growth and yield in the process according to the invention.
[0197] Nucleic acid molecules, which are advantageous for the
process according to the invention and which encode polypeptides
with the biological activity represented by the protein of the
invention and/or conferring the yield increase can be determined
from generally accessible databases. Those, which must be
mentioned, in particular in this context are general gene databases
such as the EMBL database (Stoesser G. et al., Nucleic Acids Res
2001, Vol. 29, 17-21), the GenBank database (Benson D. A. et al.,
Nucleic Acids Res 2000, Vol. 28, 15-18), or the PIR database
(Barker W. C. et al., Nucleic Acids Res. 1999, Vol. 27, 39-43). It
is furthermore possible to use organism-specific gene databases for
determining advantageous sequences, in the case of yeast for
example advantageously the SGD database (Chemy J. M. et al.,
Nucleic Acids Res. 1998, Vol. 26, 73-80) or the MIPS database
(Mewes H. W. et al., Nucleic Acids Res. 1999, Vol. 27, 44-48), in
the case of E. coli the GenProtEC database (webpage at
web.bham.ac.uk/bcm4ght6/res.html), and in the case of Arabidopsis
the TAIR-database (Huala, E. et al., Nucleic Acids Res. 2001 Vol.
29(1), 102-5) or the MIPS database.
[0198] The nucleic acid molecules used in the process according to
the invention take the form of isolated nucleic acid sequences,
which encode polypeptides with the biological activity of the
protein of the invention enabling the yield increase by reducing,
decreasing or deleting their activity. The nucleic acid sequence(s)
used in the process for yield increase in transgenic organisms
originate advantageously from an eukaryote but may also originate
from a prokaryote or an archebacterium, thus it can be derived from
e.g. a microorganism, an animal or a plant.
[0199] For the purposes of the invention, as a rule the plural is
intended to encompass the singular and vice versa.
[0200] In order to improve the introduction of the nucleic acid
sequences and the reduction, decrease or deletion of the expression
of the sequences in the transgenic organisms, which are used in the
process, the nucleic acid sequences are incorporated into a nucleic
acid construct and/or a vector in such a manner that they are
reduced, decreased or deleted in respect to the biological activity
either on the nucleic acid sequence expression level or on the
level of the polypeptide or protein encoded by said sequences. In
addition to the herein described sequences which are used in the
process according to the invention, further nucleic acid sequences,
advantageously of genes coding for oligopeptide transporter
proteins according to the invention, may additionally be present in
the nucleic acid construct or in the vector and may be introduced
into the plant in order to reduce the expression of the
polynucleotides of the invention. However, these additional
sequences may also be introduced into the plant via other, separate
nucleic acid constructs or vectors.
[0201] Using the herein mentioned cloning vectors and
transformation methods such as those which are published and cited
in: Plant Molecular Biology and Biotechnology (CRC Press, Boca
Raton, Fla.), chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors
for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1,
Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press,
1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in:
Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung
and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu Rev.
Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)) and further
cited below, the nucleic acids may be used for the recombinant
modification of a wide range of organisms, in particular
prokaryotic or eukaryotic microorganisms or plants, so their yield
is increased.
[0202] In one embodiment, the nucleic acid molecule according to
the invention originates from a plant, such as a plant selected
from the families Aceraceae, Anacardiaceae, Apiaceae, Asteraceae,
Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae,
Malvaceae, Nymphaeaceae, Papaveraceae, Rosaceae, Salicaceae,
Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae, Iridaceae,
Liliaceae, Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae,
Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae,
Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae or
Poaceae and preferably from a plant selected from the group of the
families Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae,
Fabaceae, Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae.
Preferred are crop plants and in particular plants mentioned herein
above as host plants such as the families and genera mentioned
above for example preferred the species Anacardium occidentale,
Calendula officinalis, Carthamus tinctorius, Cichorium intybus,
Cynara scolymus, Helianthus annus, Tagetes lucida, Tagetes erecta,
Tagetes tenuifolia; Daucus carota; Corylus avellana, Corylus
colurna, Borago officinalis; Brassica napus, Brassica rapa ssp.,
Sinapis arvensis, Brassica juncea, Brassica juncea var. juncea,
Brassica juncea var. crispifolia, Brassica juncea var. foliosa,
Brassica nigra, Brassica sinapioides, Melanosinapis communis,
Brassica oleracea, Arabidopsis thaliana, Anana comosus, Ananas
ananas, Bromelia comosa, Carica papaya, Cannabis sative, Ipomoea
batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulus
tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba,
Convolvulus panduratus, Beta vulgaris, Beta vulgaris var.
altissima, Beta vulgaris var. vulgaris, Beta marltima, Beta
vulgaris var. perennis, Beta vulgaris var. conditiva, Beta vulgaris
var. esculenta, Cucurbita maxima, Cucurbita mixta, Cucurbita pepo,
Cucurbita moschata, Olea europaea, Manihot utilissima, Janipha
manihot, Jatropha manihot, Manihot aipil, Manihot dulcis, Manihot
manihot, Manihot melanobasis, Manihot esculenta, Ricinus communis,
Pisum sativum, Pisum arvense, Pisum humile, Medicago sativa,
Medicago falcata, Medicago varia, Glycine max, Dolichos soja,
Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida,
Soja max, Cocos nucifera, Pelargonium grossularioides, Oleum
cocoas, Laurus nobilis, Persea americana, Arachis hypogaea, Linum
usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum
angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum,
Adenolinum grandiflorwn, Linum lewisii, Linum narbonense, Linum
perenne, Linum perenne var. lewisii, Linum pratense, Linum
trigynum, Punica granatum, Gossypium hirsutum, Gossypium arboreum,
Gossypium barbadense, Gossypium herbaceum, Gossypium thurberi, Musa
nana, Musa acuminata, Musa paradisiaca, Musa spp., Elaeis
guineensis, Papaver orientale, Papaver rhoeas, Papaver dubium,
Sesarnum indicum, Piper aduncum, Piper amalago, Piper
angustifolium, Piper auritum, Piper betel, Piper cubeba, Piper
longum, Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe
elongata, Peperomia elongata, Piper elongatum, Steffensia elongata,
Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum
secalinum, Hordeum distichon Hordeum aegiceras, Hordeum
hexastichon, Hordeum hexastichum, Hordeum irregulare, Hordeum
sativum, Hordeum secalinum, Avena saliva, Avena fatua, Avena
byzantina, Avena fatua var. sativa, Avena hybrida, Sorghum bicolor,
Sorghum halepense, Sorghum saccharatum, Sorghum vulgare, Andropogon
drummondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum,
Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum, Sorghum
dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,
Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum
subglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcus
halepensis, Sorghum miliaceum millet, Panicum militaceum, Zea mays,
Triticum aestivum, Triticum durum, Triticum turgidum, Triticum
hybernum, Triticum macha, Triticum sativum or Triticum vulgare,
Cofea spp., Coffea arabica, Coffea canephora, Coffea liberica,
Capsicum annuum, Capsicum annuum var. glabriusculum, Capsicum
frutescens, Capsicum annuum, Nicotiana tabacum, Solanum tuberosum,
Solanum melongena, Lycopersicon esculentum, Lycopersicon
lycopersicum, Lycopersicon pyriforme, Solanum integrifolium,
Solanum lycopersicum, Theobroma cacao or Camellia sinensis.
[0203] However, it is also possible to use artificial sequences,
which differ preferably in one or more bases from the nucleic acid
sequences as characterized in the invention or in one or more amino
acid molecules from polypeptide sequences as characterized in the
invention, and which mediate a polypeptide having above-mentioned
activity, e.g. having the biological activity of the protein of the
invention or conferring the yield increase after reducing,
decreasing or deleting its expression or activity.
[0204] In the process according to the invention nucleic acid
sequences can be used, which if appropriate, contain synthetic,
non-natural or modified nucleotide bases, which can be incorporated
into DNA or RNA. Said synthetic, non-natural or modified bases can
for example increase the stability of the nucleic acid molecule
outside or inside a cell. The nucleic acid molecules of the
invention can contain the same modifications as aforementioned.
[0205] As used in the present context the term "nucleic acid
molecule" may also encompass the untranslated sequence located at
the 3' and at the 5' end of the coding gene region, for example at
least 500, preferably 200, especially preferably 100, nucleotides
of the sequence upstream of the 5' end of the coding region and at
least 100, preferably 50, especially preferably 20, nucleotides of
the sequence downstream of the 3' end of the coding gene region. It
is often advantageous only to choose the coding region for cloning
and expression purposes. In the event for example the RNAi or
antisense technology is used also the 5'- and/or 3'-regions can
advantageously be used.
[0206] Preferably, the nucleic acid molecule used in the process
according to the invention or the nucleic acid molecule of the
invention is an isolated nucleic acid molecule.
[0207] An "isolated" polynucleotide or nucleic acid molecule is
separated from other polynucleotides or nucleic acid molecules,
which are present in the natural source of the nucleic acid
molecule. An isolated nucleic acid molecule may be a chromosomal
fragment of several kb, or preferably, a molecule only comprising
the coding region of the gene. Accordingly, an isolated nucleic
acid molecule of the invention may comprise chromosomal regions,
which are adjacent 5' and 3' or further adjacent chromosomal
regions, but preferably comprises no such sequences which naturally
flank the nucleic acid molecule sequence in the genomic or
chromosomal context in the organism from which the nucleic acid
molecule originates (for example sequences which are adjacent to
the regions encoding the 5'- and 3'-UTRs of the nucleic acid
molecule). In various embodiments, the isolated nucleic acid
molecule used in the process according to the invention may, for
example comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb nucleotide sequences which naturally flank the
nucleic acid molecule in the genomic DNA of the cell from which the
nucleic acid molecule originates.
[0208] The nucleic acid molecules used in the process, for example
a nucleic acid molecule with a nucleotide sequence of SEQ ID NO: 1
or SEQ ID NO: 113 or of a part thereof can be isolated using
molecular-biological standard techniques and the sequence
information provided herein. Also, for example a homologous
sequence or homologous, conserved sequence regions at the DNA or
amino acid level can be identified with the aid of comparison
algorithms. The former can be used as hybridization probes under
standard hybridization techniques (for example those described in
Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989) for isolating further nucleic acid
sequences useful in this process. A nucleic acid molecule
encompassing a complete sequence of SEQ ID NO: 1 or a part thereof
may additionally be isolated by polymerase chain reaction,
oligonucleotide primers based on this sequence or on parts thereof
being used. For example, a nucleic acid molecule comprising the
complete sequence or part thereof can be isolated by polymerase
chain reaction using oligonucleotide primers which have been
generated on the basis of this very sequence. For example, mRNA can
be isolated from cells [for example by means of the guanidinium
thiocyanate extraction method of Chirgwin et al. (1979)
Biochemistry 18:5294-5299] and cDNA can be generated by means of
reverse transcriptase (for example Moloney MLV reverse
transcriptase, available from Gibco/BRL, Bethesda, Md., or AMV
reverse transcriptase, obtainable from Seikagaku America, Inc., St.
Petersburg, Fla.). Synthetic oligonucleotide primers for the
amplification by means of polymerase chain reaction can be
generated on the basis of a sequence shown herein, for example the
sequence shown in SEQ ID NO: 1 or the sequences derived from SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ
ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:
19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ
ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:
37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ
ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:
55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ
ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:
73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ
ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID
NO: 107, SEQ ID NO: 109, SEQ ID NO: 111 or the sequences derived
from SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287.
[0209] Such primers can be used to amplify nucleic acids sequences
for example from cDNA libaries or from genomic libraries and
identify nucleic acid molecules, which are useful in the inventive
process and which have the biological activity represented by the
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288.
[0210] Advantageously the primers as depicted in SEQ ID NO: 92 and
93 are used--see table 1.
TABLE-US-00001 TABLE 1 Preferred primers SEQ Primer name ID NO:
Sequence Length_F/R at5g64410start 92 ATggCCACCgCCgACg forward
AATTCT at5g64410stop 93 TTATTTAACCggACA reverse ACCATCAACA
[0211] Moreover, it is possible to identify conserved regions from
various organisms by carrying out protein sequence alignments with
the polypeptide of the invention, in particular with the sequences
shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110 or SEQ ID NO: 112 from
which conserved regions, and in turn, degenerate primers can be
derived. Such a conserved region for the polypeptide of the
invention, is shown in SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,
SEQ ID NO: 90 and SEQ ID NO: 91. Degenerate primers can then be
utilized by PCR for the amplification of fragments of novel coding
regions coding for proteins having above-mentioned activity, e.g.
conferring the increase of the yield after reducing, decreasing or
deleting the expression or activity of the respective nucleic acid
sequence or the protein encoded by said sequence, which having the
biological activity of the protein of the invention or further
functional homologs of the polypeptide of the invention from other
organisms.
[0212] Moreover, it is possible to identify conserved regions from
various organisms by carrying out protein sequence alignments with
the polypeptide of the invention, in particular with the sequences
shown in SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 from which conserved regions, and in turn, degenerate primers
can be derived. Such a conserved region for the polypeptide of the
invention is shown in SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO:
267 and SEQ ID NO: 268. Degenerate primers can then be utilized by
PCR for the amplification of fragments of novel coding regions
coding for proteins having above-mentioned activity, e.g.
conferring the increase in yield after reducing, decreasing or
deleting the expression or activity of the respective nucleic acid
sequence or the protein encoded by said sequence, which having the
biological activity of the protein of the invention or further
functional homologs of the polypeptide of the invention from other
organisms.
[0213] These fragments can then be utilized as hybridization probe
for isolating the complete gene sequence. As an alternative, the
missing 5' and 3' sequences can be isolated by means of RACE-PCR. A
nucleic acid molecule according to the invention can be amplified
using cDNA or, as an alternative, genomic DNA as template and
suitable oligonucleotide primers, following standard PCR
amplification techniques. The nucleic acid molecule amplified thus
can be cloned into a suitable vector and characterized by means of
DNA sequence analysis. Oligonucleotides, which correspond to one of
the nucleic acid molecules used in the process, can be generated by
standard synthesis methods, for example using an automatic DNA
synthesizer.
[0214] Nucleic acid molecules which are advantageously for the
process according to the invention can be isolated based on their
homology to the nucleic acid molecules disclosed herein using the
sequences or part thereof as hybridization probe and following
standard hybridization techniques under stringent hybridization
conditions. In this context, it is possible to use, for example,
isolated nucleic acid molecules of at least 15, 20, 25, 30, 35, 40,
50, 60 or more nucleotides, preferably of at least 15, 20 or 25
nucleotides in length which hybridize under stringent conditions
with the above-described nucleic acid molecules, in particular with
those which encompass a nucleotide sequence of SEQ ID NO: 1 or SEQ
ID NO: 113. Nucleic acid molecules with 30, 50, 100, 250 or more
nucleotides may also be used.
[0215] The term "homology" means that the respective nucleic acid
molecules or encoded proteins are functionally and/or structurally
equivalent. The nucleic acid molecules that are homologous to the
nucleic acid molecules described above and that are derivatives of
said nucleic acid molecules are, for example, variations of said
nucleic acid molecules which represent modifications having the
same biological function, in particular encoding proteins with the
same or substantially the same biological function. They may be
naturally occurring variations, such as sequences from other plant
varieties or species, or mutations. These mutations may occur
naturally or may be obtained by mutagenesis techniques. The allelic
variations may be naturally occurring allelic variants as well as
synthetically produced or genetically engineered variants.
Structurally equivalents can for example be identified by testing
the binding of said polypeptide to antibodies or computer based
predictions. Structurally equivalent have the similar immunological
characteristic, e.g. comprise similar epitopes.
[0216] By "hybridizing" it is meant that such nucleic acid
molecules hybridize under conventional hybridization conditions,
preferably under stringent conditions such as described by, e.g.,
Sambrook (Molecular Cloning; A Laboratory Manual, 2.sup.nd Edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989)) or in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6.
[0217] According to the invention, DNA as well as RNA molecules of
the nucleic acid of the invention can be used as probes. Further,
as template for the identification of functional homologues
Northern blot assays as well as Southern blot assays can be
performed. The Northern blot assay advantageously provides further
information about the expressed gene product: e.g. expression
pattern, occurance of processing steps, like splicing and capping,
etc. The Southern blot assay provides additional information about
the chromosomal localization and organization of the gene encoding
the nucleic acid molecule of the invention.
[0218] A preferred, nonlimiting example of stringent Southern blot
hydridization conditions are hybridizations in 6.times. sodium
chloride/sodium citrate (.dbd.SSC) at approximately 45.degree. C.,
followed by one or more wash steps in 0.2.times.SSC, 0.1% SDS at 50
to 65.degree. C., for example at 50.degree. C., 55.degree. C. or
60.degree. C. The skilled worker knows that these hybridization
conditions differ as a function of the type of the nucleic acid
and, for example when organic solvents are present, with regard to
the temperature and concentration of the buffer. The temperature
under "standard hybridization conditions" differs for example as a
function of the type of the nucleic acid between 42.degree. C. and
58.degree. C., preferably between 45.degree. C. and 50.degree. C.
in an aqueous buffer with a concentration of 0.1.times.0.5.times.,
1.times., 2.times., 3.times., 4.times. or 5.times.SSC (pH 7.2). If
organic solvent(s) is/are present in the abovementioned buffer, for
example 50% formamide, the temperature under standard conditions is
approximately 40.degree. C., 42.degree. C. or 45.degree. C. The
hybridization conditions for DNA:DNA hybrids are preferably for
example 0.1.times.SSC and 20.degree. C., 25.degree. C., 30.degree.
C., 35.degree. C., 40.degree. C. or 45.degree. C., preferably
between 30.degree. C. and 45.degree. C. The hybridization
conditions for DNA: RNA hybrids are preferably for example
0.1.times.SSC and 30.degree. C., 35.degree. C., 40.degree. C.,
45.degree. C., 50.degree. C. or 55.degree. C., preferably between
45.degree. C. and 55.degree. C. The abovementioned hybridization
temperatures are determined for example for a nucleic acid
approximately 100 bp (=base pairs) in length and a G+C content of
50% in the absence of formamide. The skilled worker knows to
determine the hybridization conditions required with the aid of
textbooks, for example the ones mentioned above, or from the
following textbooks: Sambrook et al., "Molecular Cloning", Cold
Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985,
"Nucleic Acids Hybridization: A Practical Approach", IRL Press at
Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential
Molecular Biology: A Practical Approach", IRL Press at Oxford
University Press, Oxford.
[0219] A further example of one such stringent hybridization
condition is hybridization at 4.times.SSC at 65.degree. C.,
followed by a washing in 0.1.times.SSC at 65.degree. C. for one
hour. Alternatively, an exemplary stringent hybridization condition
is in 50% formamide, 4.times.SSC at 42.degree. C. Further, the
conditions during the wash step can be selected from the range of
conditions delimited by low-stringency conditions (approximately
2.times.SSC at 50.degree. C.) and high-stringency conditions
(approximately 0.2.times.SSC at 50.degree. C., preferably at
65.degree. C.) (20.times.SSC: 0.3M sodium citrate, 3M NaCl, pH
7.0). In addition, the temperature during the wash step can be
raised from low-stringency conditions at room temperature,
approximately 22.degree. C., to higher-stringency conditions at
approximately 65.degree. C. Both of the parameters salt
concentration and temperature can be varied simultaneously, or else
one of the two parameters can be kept constant while only the other
is varied. Denaturants, for example formamide or SDS, may also be
employed during the hybridization. In the presence of 50%
formamide, hybridization is preferably effected at 42.degree. C.
Relevant factors like i) length of treatment, ii) salt conditions,
iii) detergent conditions, iv) competitor DNAs, v) temperature and
vi) probe selection can combined case by case so that not all
possibilities can be mentioned herein.
[0220] Some examples of conditions for DNA hybridization (Southern
blot assays) and wash step are shown below:
(1) Hybridization conditions can be selected, for example, from the
following conditions: [0221] a) 4.times.SSC at 65.degree. C.,
[0222] b) 6.times.SSC at 45.degree. C., [0223] c) 6.times.SSC, 100
mg/ml denatured fragmented fish sperm DNA at 68.degree. C., [0224]
d) 6.times.SSC, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at
68.degree. C., [0225] e) 6.times.SSC, 0.5% SDS, 100 mg/ml denatured
fragmented salmon sperm DNA, 50% formamide at 42.degree. C., [0226]
f) 50% formamide, 4.times.SSC at 42.degree. C., [0227] g) 50%
(vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%
poly-vinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM
NaCl, 75 mM sodium citrate at 42.degree. C., [0228] h) 2.times. or
4.times.SSC at 50.degree. C. (low-stringency condition), or [0229]
i) 30 to 40% formamide, 2.times. or 4.times.SSC at 42.degree. C.
(low-stringency condition). (2) Wash steps can be selected, for
example, from the following conditions: [0230] a) 0.015 M
NaCl/0.0015 M sodium citrate/0.1% SDS at 50.degree. C. [0231] b)
0.1.times.SSC at 65.degree. C. [0232] c) 0.1.times.SSC, 0.5% SDS at
68.degree. C. [0233] d) 0.1.times.SSC, 0.5% SDS, 50% formamide at
42.degree. C. [0234] e) 0.2.times.SSC, 0.1% SDS at 42.degree. C.
[0235] f) 2.times.SSC at 65.degree. C. (low-stringency condition).
[0236] g) 0.2.times.SSC, 0.1% SDS at 60.degree. C. (medium-high
stringency conditions), or [0237] h) 0.1.times.SSC, 0.1% SDS at
60.degree. C. (medium-high stringency conditions), or [0238] I)
0.2.times.SSC, 0.1% SDS at 65.degree. C. (high stringency
conditions), or [0239] h) 0.1.times.SSC, 0.1% SDS at 65.degree. C.
(high stringency conditions)
[0240] Polypeptides having above-mentioned activity, e.g.
conferring the yield increase, derived from other organisms, can be
encoded by other DNA sequences, which hybridize to the sequences
shown in SEQ ID NO: 1 or SEQ ID NO: 113 under relaxed hybridization
conditions and which code on expression for peptides having the
further biological activities of the protein of the invention.
[0241] Further, some applications have to be performed at low
stringency hybridization conditions, without any consequences for
the specificity of the hybridization. For example, a Southern blot
analysis of total DNA could be probed with a nucleic acid molecule
of the present invention and washed at low stringency (55.degree.
C. in 2.times.SSPE, 0.1% SDS). The hybridisation analysis could
reveal a simple pattern of only genes encoding polypeptides of the
present invention, e.g. having herein-mentioned yield increasing
activity and/or having also the biological activity of an
oligopeptide transporter protein as used in the invention. A
further example of such low-stringent hybridization conditions is
4.times.SSC at 50.degree. C. or hybridization with 30 to 40%
formamide at 42.degree. C. Such molecules comprise those which are
fragments, analogues or derivatives of the polypeptide of the
invention and differ, for example, by way of amino acid and/or
nucleotide deletion(s), insertion(s), substitution (s), addition(s)
and/or recombination (s) or any other modification(s) known in the
art either alone or in combination from the above-described amino
acid sequences or their underlying nucleotide sequence(s). However,
it is preferred to use high stringency hybridisation
conditions.
[0242] Hybridization should advantageously be carried out with
fragments of at least 5, 10, 15, 20, 25, 30, 35 or 40 bp,
advantageously at least 50, 60, 70 or 80 bp, preferably at least
90, 100 or 110 bp. Most preferably are fragments of at least 15,
20, 25 or 30 bp. Preferably are also hybridizations with at least
100 bp or 200, very especially preferably at least 400 bp in
length. In an especially preferred embodiment, the hybridization
should be carried out with the entire nucleic acid sequence with
conditions described above.
[0243] The terms "fragment", "fragment of a sequence" or "part of a
sequence" mean a truncated sequence of the original sequence
referred to. The truncated sequence (nucleic acid or protein
sequence) can vary widely in length; the minimum size being a
sequence of sufficient size to provide a sequence or sequence
fragment with at least 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30 bp in length with at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or
99% identity preferably 100% identity with a fragment of the
nucleic acid molecules of the invention for example with the
sequences shown in SEQ ID NO: 1 or SEQ ID NO: 113. Said truncated
sequences can as mentioned vary widely in length from 15 bp up to 2
kb or more, advantageously the sequences have a minimal length of
15, 20, 25, 30, 35 or 40 bp, while the maximum size is not
critical. 100, 200, 300, 400, 500 or more base pair fragments can
be used. In some applications, the maximum size usually is not
substantially greater than that required to provide the complete
gene function(s) of the nucleic acid sequences of the invention.
Such sequences can advantageously been used for the repression,
reduction, decrease or deletion of the biological activity of the
nucleic acid molecules and/or proteins of the invention by for
example the RNAi- or antisense-technology. For the reduction,
decrease or deletion of the biological activity of the inventive
nucleic acid sequence and/or the inventive protein also the
promotor regions of the disclosed nucleic acid sequences can be
used. The skilled worker knows how to clone said promotor
regions.
[0244] Typically, the truncated amino acid sequence will range from
about 5 to about 310 amino acids in length. More typically,
however, the sequence will be a maximum of about 250 amino acids in
length, preferably a maximum of about 200 or 100 amino acids. It is
usually desirable to select sequences of at least about 10, 12 or
15 amino acids, up to a maximum of about 20 or 25 amino acids.
[0245] The term "epitope" relates to specific immunoreactive sites
within an antigen, also known as antigenic determinates. These
epitopes can be a linear array of monomers in a polymeric
composition--such as amino acids in a protein--or consist of or
comprise a more complex secondary or tertiary structure. Those of
skill will recognize that immunogens (i.e., substances capable of
eliciting an immune response) are antigens; however, some antigen,
such as haptens, are not immunogens but may be made immunogenic by
coupling to a carrier molecule. The term "antigen" includes
references to a substance to which an antibody can be generated
and/or to which the antibody is specifically immunoreactive.
[0246] In one embodiment the present invention relates to an
epitope of the poly-peptide of the present invention.
[0247] The term "one or several amino acids" relates to at least
one amino acid but not more than that number of amino acids, which
would result in a homology of below 50% identity. Preferably, the
identity is more than 70% or 80%, more preferred are 85%, 90%, 91%,
92%, 93%, 94% or 95%, even more preferred are 96%, 97%, 98%, or 99%
identity.
[0248] Further, the nucleic acid molecule of the invention
comprises a nucleic acid molecule, which is a complement of one of
the nucleotide sequences of above mentioned nucleic acid molecules
or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences shown in SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ
ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:
19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ
ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:
37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ
ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:
55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ
ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:
73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ
ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID
NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:
115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:
123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:
187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:
195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:
203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:
211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:
219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:
227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:
235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:
243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO:
251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO:
275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO:
283, SEQ ID NO: 285 or SEQ ID NO: 287 is one which is sufficiently
complementary to one of the nucleotide sequences shown in SEQ ID
NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ
ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ. ID NO: 17, SEQ ID NO:
19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ
ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:
37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ
ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:
55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ
ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:
73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ
ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID
NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:
115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:
123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:
187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:
195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:
203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:
211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:
219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:
227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:
235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:
243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO:
251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO:
275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO:
283, SEQ ID NO: 285 or SEQ ID NO: 287 such that it can hybridize to
one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,
SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID
NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57,
SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID
NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75,
SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID
NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO:
109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:
117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO:
125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO:
133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO:
141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO:
149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:
157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO:
165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:
173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:
181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO:
189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO:
197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO:
205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO:
213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO:
221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO:
229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO:
237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO:
245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO:
269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO:
277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285
or SEQ ID NO: 287 thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybrization conditions.
However, a complement of one of the herein disclosed sequences is
preferably a sequence complement thereto according to the base
pairing of nucleic acid molecules well known to the skilled person.
For example, the bases A and G undergo base pairing with the bases
T and U or C, resp. and vice versa. Modifications of the bases can
influence the base-pairing partner.
[0249] The nucleic acid molecule of the invention comprises a
nucleotide sequence which is at least about 30%, 35%, 40% or 45%,
preferably at least about 50%, 55%, 60% or 65%, more preferably at
least about 70%, 80%, or 90%, and even more preferably at least
about 95%, 97%, 98%, 99% or more homologous to a nucleotide
sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID
NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID
NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID
NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,
SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID
NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:
103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:
111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO.: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 or a portion thereof and/or has the biological activity of the
protein of the invention or the nucleic acid molecule encoding said
protein. The nucleic acid molecule of the invention comprises a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions as defined herein, to one of the nucleotide
sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID
NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID
NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID
NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,
SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID
NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:
103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:
111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 or a portion thereof and encodes a protein having
aforementioned activity, e.g. conferring a yield increase upon the
reduction of deletion of its activity, and optionally, the
biological activity of the protein of the invention.
[0250] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the coding region of one of the
sequences in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ
ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:
25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ
ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:
61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ
ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO:
79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103,
SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ
ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID
NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287 for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the nucleic acid molecule
or polypeptide of the present invention or a fragment encoding a
non active part of the nucleic acid molecule or the polypeptide of
the invention, i.e. having abovementioned activity, e.g. conferring
an increase in yield if its expression or activity is decreased.
The nucleotide sequences determined from the cloning of the present
protein according to the invention encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., in SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ
ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:
31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ
ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ
ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO:
67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ
ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:
85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109,
SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ
ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID
NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 an anti-sense sequence of one of the sequences, e.g., set forth
in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,
SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID
NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,
SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID
NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,
SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID
NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71,
SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID
NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO:
105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO:
113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287 or naturally
occurring mutants thereof. Primers based on a nucleotide of
invention can be used in PCR reactions to clone homologues of the
polypeptide of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples.
Said nucleic acid molecules, which are homologues of the sequence
of the invention or the nucleic acid molecules of the invention
themselves can be used to reduce, decrease or delete the biological
activity of the nucleic acid molecules and/or proteins of the
invention.
[0251] Primer sets are interchangable. The person skilled in the
art knows to combine said primers to result in the desired product,
e.g. in a full-length clone or a partial sequence. Probes based on
the sequences of the nucleic acid molecule of the invention can be
used to detect transcripts or genomic sequences encoding the same
or homologous proteins. The probe can further comprise a label
group attached thereto, e.g. the label group can be a
radio-isotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a genomic marker
test kit for identifying cells which express or does not express a
polypepetide of the invention, such as by measuring a level of an
encoding nucleic acid molecule in a sample of cells, e.g.,
detecting mRNA levels or determining, whether a genomic gene
comprising the sequence of the polynucleotide of the invention has
been mutated or deleted.
[0252] The nucleic acid molecule of the invention encodes a
polypeptide or portion thereof which includes an amino acid
sequence which is sufficiently homologous to the amino acid
sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 such that the
protein or portion thereof maintains the ability to participate in
the yield increase, in particular a compound such as the protein of
the invention which is involved in the reduction, decrease and/or
deletion of the yield increase by for example metabolization,
degradation or export of undesired chemical compounds and thereby
increasing the yield upon the reduction or deletion of its activity
as mentioned above or as described in the examples in plants is
comprised.
[0253] As used herein, the language "sufficiently homologous"
refers to proteins or portions thereof which have amino acid
sequences which include a minimum number of identical or equivalent
amino acid residues (e.g., an amino acid residue which has a
similar side chain as an amino acid residue in one of the sequences
of the polypeptide of the present invention) to an amino acid
sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID
NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID
NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,
SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID
NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,
SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID
NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO:
104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288. Portions of the aforementioned amino acid sequence are at
least 3, 5, 10, 20, 30, 40, 50 or more amino acids in length.
Nucleic acid sequences, which as a result of the degeneracy of the
genetic code, can be derived from said polypeptide sequences can be
used for the repression, decrease or deletion of the biological
activity of the polypeptide or the nucleic acid molecule of the
invention according to the disclosure herein.
[0254] In one embodiment, the nucleic acid molecule of the present
invention comprises a nucleic acid that encodes a portion of the
protein of the present invention. The protein is at least about
30%, 35%, 40%, 45% or 50%, preferably at least about 55%, 60%, 65%
or 70% and more preferably at least about 75%, 80%, 85%, 90%, 91%,
92%, 93% or 94% and most preferably at least about 95%, 97%, 98%,
99% or more homologous to an entire amino acid sequence of SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:
20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ
ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:
38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ
ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:
56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ
ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:
74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ
ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID
NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288 and having above-mentioned
activity, e.g. conferring preferably the increase in yield upon
being reduced in its activity.
[0255] Portions of proteins encoded by the nucleic acid molecule of
the invention are preferably in such a manner biologically active,
that they are increasing the yield of plant growth by being in its
biological activity reduced, decreased or deleted.
[0256] As mentioned herein, the term "biologically active portion"
is intended to include a portion, e.g., a domain/motif, that
confers by introducing said nucleic acid sequence or part thereof
an increase of the yield in plant growth.
[0257] The invention further relates to nucleic acid molecules
which are used in the inventive process and which as a result of
degeneracy of the genetic code can be derived from a polypeptide
sequence as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 and thus encoding a polypeptide of the present invention, in
particular a polypeptide leading by reducing, decreasing or
deleting its biological activity to an increase of the yield of
plant growth. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, an amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288 which differ from said amino acid sequences in at
least one or more amino acids.
[0258] In addition, it will be appreciated by those skilled in the
art that DNA sequence polymorphisms that lead to changes in the
amino acid sequences may exist within a population. Such genetic
polymorphism in the gene encoding the polypeptide of the invention
or comprising the nucleic acid molecule of the invention may exist
among individuals within a population due to natural variation.
[0259] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
encoding the polypeptide of the invention or comprising the nucleic
acid molecule of the invention, preferably encoding the polypeptide
of the invention or comprising the nucleic acid molecule of the
invention derived from a crop plant or from a microorganism useful
for increasing the yield in plants upon reduction in its activity,
in particular encoding a oligopeptide transporter protein of the
invention or comprising the nucleic acid molecule of the invention
derived from a crop plant or a microorganism for increasing the
growth of plants or plant parts upon reduction of its activity.
Such natural variations can typically result in 1-5% variance in
the nucleotide sequence of the gene used in the inventive process.
Any and all such nucleotide variations and resulting amino acid
polymorphisms in genes encoding a polypeptide of the invention or
comprising the nucleic acid molecule of the invention that are the
result of natural variation and that in the event of reducing,
decreasing or deleting its biological activity do not alter the
functional activity as described are intended to be within the
scope of the invention.
[0260] Nucleic acid molecules corresponding to natural variants
homologues of a nucleic acid molecule of the invention, which can
also be a cDNA, can be isolated based on their homology to the
nucleic acid molecules disclosed herein using the nucleic acid
molecule of the invention, or a portion thereof, as a hybridization
probe according to standard hybridization techniques under
stringent hybridization conditions.
[0261] Accordingly, in another embodiment, a nucleic acid molecule
of the invention is at least 15, 20, 25 or 30 nucleotides in
length. Preferably, it hybridizes under stringent conditions to a
nucleic acid molecule comprising a nucleotide sequence of the
nucleic acid molecule of the present invention, e.g. comprising the
sequence shown in SEQ ID NO: 1 or SEQ ID NO: 113. The nucleic acid
molecule is preferably at least 20, 30, 50, 100, 250 or more
nucleotides in length.
[0262] The term "hybridizes under stringent conditions" is defined
above. In one embodiment, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences of at least 30%, 40%,
50% or 65% identical to each other typically remain hybridized to
each other. Preferably, the conditions are such that sequences of
at least about 70%, more preferably at least about 75% or 80%, and
even more preferably of at least about 85%, 90% or 95% or more
identical to each other typically remain hybridized to each
other.
[0263] Preferably, nucleic acid molecule of the invention that
hybridizes under stringent conditions to a sequence of SEQ ID NO: 1
or SEQ ID NO: 113 corresponds to a naturally-occurring nucleic acid
molecule of the invention. As used herein, a "naturally-occurring"
nucleic acid molecule refers to a RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein). Preferably, the nucleic acid molecule encodes a natural
protein having above-mentioned activity, e.g. conferring increase
in plant growth or plant parts after reducing, decreasing or
deleting the expression or activity thereof or the activity of a
protein having the biological activity of the protein of the
invention.
[0264] In addition to naturally-occurring variants of the nucleic
acid or protein sequence of the invention that may exist in the
population, the skilled artisan will further appreciate that
changes can be introduced by mutation into a nucleotide sequence of
the nucleic acid molecule encoding the polypeptide of the
invention, thereby leading to changes in the amino acid sequence of
the encoded polypeptide of the invention and thereby altering the
functional ability of the polypeptide, meaning preferably reducing,
decreasing or deleting said activity. For example, nucleotide
substitutions leading to amino acid substitutions at "essential"
amino acid residues can be made in a sequence of the nucleic acid
molecule of the invention, e.g. in SEQ ID NO: 1 or SEQ ID NO: 113.
An "essential" amino acid residue is a residue that if altered from
the wild-type sequence of one of the polypeptide of the invention
lead to an altered activity of said polypeptide, whereas a
"non-essential" amino acid residue is not required for the activity
of the protein for example for the activity as an enzyme. The
alteration of "essential" residues lead to a reduced decreased or
deleted activity of the polypeptides of the invention. Preferably
amino acids of the polypeptide of the invention are change in such
a manner that the activity is reduced, decreased or deleted that
means preferably essential amino acid residues and/or more
non-essential residues are changed and thereby the activity is
reduced, which leads as mentioned above to an increase in growth of
plants or plant parts after decreasing the expression or activity
of the polypeptide of the invention. Other amino acid residues,
however, (e.g., those that are not conserved or only semi-conserved
in the domain having said activity) may not be essential for
activity and thus are likely to be amenable to alteration without
altering said activity are less preferred.
[0265] Further, a person skilled in the art knows that the codon
usage between organisms can differ. Therefore, he will adapt the
codon usage in the nucleic acid molecule of the present invention
to the usage of the organism in which the polynucleotide or
polypeptide is expressed, so that the expression of the nucleic
acid molecule or the encoded protein of the invention is more
likely reduced.
[0266] Accordingly, the invention relates to nucleic acid molecules
encoding poly-peptide having abovementioned activity, e.g.
conferring an increased growth in a plant or plant part. Such
polypeptides differ in amino acid sequence from a sequence
contained in, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:
44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ
ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:
62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ
ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:
80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104,
SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ
ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID
NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 yet do not
retain said biological activity described herein and thereby
enabling the increase in growth. The nucleic acid molecule can
comprise a nucleotide sequence encoding a polypeptide, wherein the
polypeptide comprises an amino acid sequence at least about 50%
identical to an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288 and is capable of participation in the increase
of plant growth after decreasing its expression or its biological
function. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60%, 70% or 80% identical to the
sequence in SEQ ID NO: 2 or SEQ ID NO: 114, more preferably at
least about 85% identical to SEQ ID NO: 2 or SEQ ID NO: 114, even
more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%
homologous to the sequence in SEQ ID NO: 2 or SEQ ID NO: 114, and
most preferably at least about 96%, 97%, 98%, or 99% identical to
the sequence in SEQ ID NO: 2 or SEQ ID NO: 114.
[0267] To determine the percentage homology (=identity) of two
amino acid sequences (for example of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288) or of two nucleic acid molecules (for example of
the sequence SEQ ID NO: 1 or SEQ ID NO: 3, the sequences are
written one underneath the other for an optimal comparison (for
example gaps may be inserted into the sequence of a protein or of a
nucleic acid in order to generate an optimal alignment with the
other protein or the other nucleic acid). The amino acid residues
or nucleic acid molecules at the corresponding amino acid positions
or nucleotide positions are then compared. If a position in one
sequence is occupied by the same amino acid residue or the same
nucleic acid molecule as the corresponding position in the other
sequence, the molecules are homologous at this position (i.e. amino
acid or nucleic acid "homology" as used in the present context
corresponds to amino acid or nucleic acid "identity". The
percentage homology between the two sequences is a function of the
number of identical positions shared by the sequences (i.e. %
homology=number of identical positions/total number of
positions.times.100). The terms "homology" and "identity" are thus
to be considered as synonyms for this description.
[0268] For the determination of the percentage homology (=identity)
of two or more amino acids or of two or more nucleotide sequences
several computer software programs have been developed. The
homology of two or more sequences can be calculated with for
example the software fasta, which presently has been used in the
version fasta 3 (W. R. Pearson and D. J. Lipman (1988), Improved
Tools for Biological Sequence Comparison.PNAS 85:2444-2448; W. R.
Pearson (1990) Rapid and Sensitive Sequence Comparison with FASTP
and FASTA, Methods in Enzymology 183:63-98; W. R. Pearson and D. J.
Lipman (1988) Improved Tools for Biological Sequence
Comparison.PNAS 85:2444-2448; W. R. Pearson (1990); Rapid and
Sensitive Sequence Comparison with FASTP and FASTAMethods in
Enzymology 183:63-98). Another useful program for the calculation
of homologies of different sequences is the standard blast program,
which is included in the Biomax pedant software (Biomax, Munich,
Federal Republic of Germany). This leads unfortunately sometimes to
suboptimal results since blast does not always include complete
sequences of the subject and the querry. Nevertheless as this
program is very efficient it can be used for the comparison of a
huge number of sequences. The following settings are typically used
for such a comparisons of sequences:
[0269] -p Program Name [String]; -d Database [String]; default=nr;
-i Query File [File In]; default=stdin; -e Expectation value (E)
[Real]; default=10.0; -m alignment view options: 0=pairwise;
1=query-anchored showing identities; 2=query-anchored no
identities; 3=flat query-anchored, show identities; 4=flat
query-anchored, no identities; 5=query-anchored no identities and
blunt ends; 6=flat query-anchored, no identities and blunt ends;
7=XML Blast output; 8=tabular; 9 tabular with comment lines
[Integer]; default=0; -o BLAST report Output File [File Out]
Optional; default=stdout; -F Filter query sequence (DUST with
blastn, SEG with others) [String]; default=T; -G Cost to open a gap
(zero invokes default behavior) [Integer]; default=0; -E Cost to
extend a gap (zero invokes default behavior) [Integer]; default=0;
-X X dropoff value for gapped alignment (in bits) (zero invokes
default behavior); blastn 30, megablast 20, tblastx 0, all others
15 [Integer]; default=0; -I Show GI's in defines [T/F]; default=F;
-q Penalty for a nucleotide mismatch (blastn only) [Integer];
default=-3; -r Reward for a nucleotide match (blastn only)
[Integer]; default=1; -v Number of database sequences to show
one-line descriptions for (V) [Integer]; default=500; -b Number of
database sequence to show alignments for (B) [Integer];
default=250; -f Threshold for extending hits, default if zero;
blastp 11, blastn 0, blastx 12, tblastn 13; tblastx 13, megablast 0
[Integer]; default=0; -g Perfom gapped alignment (not available
with tblastx) [T/F]; default=T; -Q Query Genetic code to use
[Integer]; default=1; -D DB Genetic code (for tblast[nx] only)
[Integer]; default=1; -a Number of processors to use [Integer];
default=1; -O SeqAlign file [File Out] Optional; -J Believe the
query define [T/F]; default=F; -M Matrix [String];
default=BLOSUM62; -W Word size, default if zero (blastn 11,
megablast 28, all others 3) [Integer]; default=0; -z Effective
length of the database (use zero for the real size) [Real];
default=0; -K Number of best hits from a region to keep (off by
default, if used a value of 100 is recommended) [Integer];
default=0; -P 0 for multiple hit, 1 for single hit [Integer];
default=0; -Y Effective length of the search space (use zero for
the real size) [Real]; default=0; -S Query strands to search
against database (for blast[nx], and tblastx); 3 is both, 1 is top,
2 is bottom [Integer]; default=3; -T Produce HTML output [T/F];
default=F; -1 Restrict search of database to list of GI's [String]
Optional; -U Use lower case filtering of FASTA sequence [T/F]
Optional; default=F; -y X dropoff value for ungapped extensions in
bits (0.0 invokes default behavior); blastn 20, megablast 10, all
others 7 [Real]; default=0.0; -Z X dropoff value for final gapped
alignment in bits (0.0 invokes default behavior); blastn/megablast
50, tblastx 0, all others 25 [Integer]; default=0; -R PSI-TBLASTN
checkpoint file [File In] Optional; -n MegaB-last search [T/F];
default=F; -L Location on query sequence [String] Optional; -A
Multiple Hits window size, default if zero (blastn/megablast 0, all
others 40 [Integer]; default=0; -w Frame shift penalty (OOF
algorithm for blastx) [Integer]; default=0; -t Length of the
largest intron allowed in tblastn for linking HSPs (0 disables
linking) [Integer]; default=0.
[0270] Results of high quality are reached by using the algorithm
of Needleman and Wunsch or Smith and Waterman. Therefore programs
based on said algorithms are preferred. Advantageously the
comparisons of sequences can be done with the program PileUp (J.
Mol. Evolution, 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989:
151-153) or preferably with the programs Gap and BestFit, which are
respectively based on the algorithms of Needleman and Wunsch [J.
Mol. Biol. 48; 443-453 (1970)] and Smith and Waterman [Adv. Appl.
Math. 2; 482-489 (1981)]. Both programs are part of the GCG
software-package [Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711 (1991); Altschul et al. (1997) Nucleic
Acids Res. 25:3389 et seq.]. Therefore preferably the calculations
to determine the perentages of sequence homology are done with the
program Gap over the whole range of the sequences. The following
standard adjustments for the comparison of nucleic acid sequences
were used: gap weight: 50, length weight: 3, average match: 10.000,
average mismatch: 0.000.
[0271] For example a sequence which has a 80% homology with
sequence SEQ ID NO: 1 at the nucleic acid level is understood as
meaning a sequence which, upon comparison with the sequence SEQ ID
NO: 1 by the above Gap program algorithm with the above parameter
set, has 80% homology.
[0272] Homology between two polypeptides is understood as meaning
the identity of the amino acid sequence over in each case the
entire sequence length which is calculated by comparison with the
aid of the program algorithm Gap (Wisconsin Package Version 10.0,
University of Wisconsin, Genetics Computer Group (GCG), Madison,
USA), setting the following parameters: gap weight: 8; length
weight: 2; average match: 2.912; average mismatch: -2.003.
[0273] For example a sequence which has a 80% homology with
sequence SEQ ID NO: 2 at the protein level is understood as meaning
a sequence which, upon comparison with the sequence SEQ ID NO: 2 by
the above Gap program algorithm with the above parameter set, has
80% homology.
[0274] Functional equivalents derived from one of the polypeptides
as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 according to
the invention by substitution, insertion or deletion have at least
30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%
by preference at least 80%, especially preferably at least 85% or
90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%,
97%, 98% or 99% homology with one of the polypeptides as shown in
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:
28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ
ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ
ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:
64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ
ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:
82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106,
SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ
ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID
NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288 according to the invention
and are characterized by essentially the same properties as the
polypeptide as shown in SEQ ID NO: 2 or SEQ ID NO: 114 of A.
thaliana.
[0275] Functional equivalents derived from the nucleic acid
sequences as shown in SEQ ID NO: 1 or SEQ ID NO: 113 according to
the invention by substitution, insertion or deletion have at least
30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%
by preference at least 80%, especially preferably at least 85% or
90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%,
97%, 98% or 99% homology with one of the polypeptides as shown in
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:
28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ
ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ
ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:
64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ
ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:
82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106,
SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 or SEQ ID NO: 114,
SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ
ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID
NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288 respectively according to the
invention and encode polypeptides having essentially the same
properties as the polypeptide as shown in SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112 or SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288.
[0276] "Essentially the same properties" of a functional equivalent
is above all understood as meaning that the functional equivalent
has above mentioned activity, e.g. conferring an increasing growth
of plants or plant parts while decreasing the amount of protein,
activity or function of said functional equivalent in a plant, in a
plant tissue, plant cells or a part of the same.
[0277] A nucleic acid molecule encoding a homologe to a protein
sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112 or SEQ ID
NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 can be
created by introducing one or more nucleotide substitutions,
additions or deletions into a nucleotide sequence of the nucleic
acid molecule of the present invention, in particular of SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19,
SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID
NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,
SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID
NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,
SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID
NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,
SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID
NO:83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO:
107, SEQ ID NO: 109, SEQ ID NO: 111 or SEQ ID NO: 113, SEQ ID NO:
115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:
123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:
187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:
195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:
203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:
211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:
219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:
227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:
235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:
243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO:
251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO:
275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO:
283, SEQ ID NO: 285 or SEQ ID NO: 287 such that one or more amino
acid substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into the sequences of,
e.g. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,
SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID
NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,
SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID
NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,
SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID
NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71,
SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID
NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO:
105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO:
113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[0278] Preferably, non-conservative amino acid substitutions are
made at one or more predicted non-essential or preferably essential
amino acid residues and thereby reducing, decreasing or deleting
the activity of the respective protein. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine).
[0279] Thus, a predicted nonessential or essential amino acid
residue in a polypeptide of the invention is preferably replaced
with another amino acid residue from another family. Alternatively,
in another embodiment, mutations can be introduced randomly along
all or part of a coding sequence of a nucleic acid molecule of the
invention, such as by saturation mutagenesis, and the resultant
mutants can be screened for activity described herein to identify
mutants that lost or have decreased biological activity, e.g.
conferring an increase in plant growth or growth of plant
parts.
[0280] Most preferably the activity of the polypeptides of the
invention can be reduced or deleted by for example creation of stop
codons through mutation or insertions.
[0281] Following mutagenesis of one of the sequences of SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19,
SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID
NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,
SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID
NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,
SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID
NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,
SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID
NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO:
107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:
115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:
123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:
187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO:
195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO:
203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO:
211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:
219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO:
227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:
235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO:
243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO:
251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO:
275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO: 281, SEQ ID NO:
283, SEQ ID NO: 285 or SEQ ID NO: 287 the encoded protein can be
expressed recombinantly and the activity of the protein can be
determined using, for example, assays described herein (see
Examples).
[0282] The highest homology of the polypeptide molecule SEQ ID NO:
2 used in the process according to the invention was found for the
following database entries by Gap search:
TABLE-US-00002 Hit Best Homolog % Identity SEQ ID NO: 2
SPTREMBL_Q6YUP9 88 SEQ ID NO: 2 SPTREMBL_Q8S2I4 87 SEQ ID NO: 2
SPTREMBL_Q7F9L4 86 SEQ ID NO: 2 PIR_F86233 84 SEQ ID NO: 2
PIR_T01900 80 SEQ ID NO: 2 SPTREMBL_Q8H641 80
[0283] Homologs of the nucleic acid sequences used, with the
sequence SEQ ID NO: 1 or of the nucleic acid sequences derived from
the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ
ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:
25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ
ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:
61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ
ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO:
79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103,
SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111
comprise also allelic variants with at least approximately 30%,
35%, 40% or 45% homology, by preference at least approximately 50%,
60% or 70%, more preferably at least approximately 90%, 91%, 92%,
93%, 94% or 95% and even more preferably at least approximately
96%, 97%, 98%, 99% or more homology with one of the nucleotide
sequences shown or the abovementioned derived nucleic acid
sequences or their homologues, derivatives or analogues or parts of
these. Allelic variants encompass in particular functional variants
which can be obtained by deletion, insertion or substitution of
nucleotides from the sequences shown, preferably from SEQ ID NO: 1,
or from the derived nucleic acid sequences, the intention being,
however, that the enzyme activity or the biological activity of the
resulting proteins synthesized is advantageously lost or
decreased.
[0284] Homologs of the nucleic acid sequences used, with the
sequence SEQ ID NO: 113 or of the nucleic acid sequences derived
from the sequences SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117,
SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ
ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID
NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO:
183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO:
191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:
199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO:
207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:
215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO:
231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO:
239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO:
247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO:
271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO:
279, SEQ ID NO: 281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO:
287 comprise also allelic variants with at least approximately 30%,
35%, 40% or 45% homology, by preference at least approximately 50%,
60% or 70%, more preferably at least approximately 90%, 91%, 92%,
93%, 94% or 95% and even more preferably at least approximately
96%, 97%, 98%, 99% or more homology with one of the nucleotide
sequences shown or the abovementioned derived nucleic acid
sequences or their homologues, derivatives or analogues or parts of
these. Allelic variants encompass in particular functional variants
which can be obtained by deletion, insertion or substitution of
nucleotides from the sequences shown, preferably from SEQ ID NO:
113, or from the derived nucleic acid sequences, the intention
being, however, that the enzyme activity or the biological activity
of the resulting proteins synthesized is advantageously lost or
decreased.
[0285] In one embodiment of the present invention, the nucleic acid
molecule comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO:
113. It is preferred that the nucleic acid molecule comprises as
little as possible other nucleotides not shown in SEQ ID NO: 1 or
SEQ ID NO: 113. In one embodiment, the nucleic acid molecule
comprises less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or
40 further nucleotides. In a further embodiment, the nucleic acid
molecule comprises less than 30, 20 or 10 further nucleotides.
[0286] Also preferred is that the nucleic acid molecule of the
invention encodes a polypeptide comprising the sequence shown in
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:
28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ
ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO:
46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ
ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:
64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ
ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:
82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106,
SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ
ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID
NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288.
[0287] In one embodiment, the nucleic acid molecule encodes less
than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids. In a
further embodiment, the encoded polypeptide comprises less than 20,
15, 10, 9, 8, 7, 6 or 5 further amino acids. In one embodiment used
in the inventive process, the encoded polypeptide is identical to
the sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288.
[0288] In one embodiment, the nucleic acid molecule encoding a
polypeptide comprising the sequence shown in SEQ ID NO: 1 or SEQ ID
NO: 113 comprises less than 100 further nucleotides. In a further
embodiment, the nucleic acid molecule comprises less than 30
further nucleotides.
[0289] Polypeptides (=proteins), which still have the
abovementioned activity of the polypeptide of the present
invention, e.g. conferring an increase of growth in plants or plant
parts or having the biological activity of an oligopeptide
transporter protein of the invention, i.e. polypeptides whose
activity is essentially reduced, are polypeptides by at least 10%
or 20%, by preference 30% or 40%, especially preferably 50% or 60%,
very especially preferably 80% or 90% or more in comparison to the
wild type biological activity or enzyme activity, advantageously,
the activity is essentially reduced in comparison with the activity
of a protein encoded by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288 and expressed under identical conditions.
[0290] Homologs of SEQ ID NO: 1 or of the derived sequences of SEQ
ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID
NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27,
SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID
NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,
SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID
NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,
SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID
NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,
SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 105, SEQ
ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111 also mean truncated
sequences, cDNA, single-stranded DNA or RNA of the coding and
noncoding DNA sequence. Homologus of SEQ ID NO: 1 or the derived
sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ
ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:
25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ
ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:
61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ
ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO:
79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103,
SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111 are
also understood as meaning derivatives which comprise noncoding
regions such as, for example, UTRs, terminators, enhancers or
promoter variants. The promoters upstream of the nucleotide
sequences stated can be modified by one or more nucleotide
substitution(s), insertion(s) and/or deletion(s) with, however,
preferably interfering with the functionality or activity either of
the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is decreased by
modification of their sequence or their regulation, or that they
are replaced completely by less active promoters and thereby the
activity of the expressed nucleic acid sequence is reduced or
deleted, even promoters from heterologous organisms. Appropriate
promoters are known to the person skilled in the art and are
mentioned herein below. Further methods exists to modulate the
promoters of the genes of the invention, e.g. by modifying the
activity of transacting factors, meaning natural or artificial
transcription factors, which can bind to the promoter and influence
its activity. Furthermore it is possible to influence promoters of
interest by modifying upstream signaling components like receptors
or kinases, which are involved in the regulation of the promoter of
interest.
[0291] Homologs of SEQ ID NO: 113 or of the derived sequences of
SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ
ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID
NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287 also mean
truncated sequences, cDNA, single-stranded DNA or RNA of the coding
and non-coding DNA sequence. Homologus of SEQ ID NO: 113 or the
derived sequences of are also understood as meaning derivatives
which comprise noncoding regions such as, for example, UTRs,
terminators, enhancers or promoter variants. The promoters upstream
of the nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s) with,
however, preferably interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is decreased by
modification of their sequence or their regulation, or that they
are replaced completely by less active promoters and thereby the
activity of the expressed nucleic acid sequence is reduced or
deleted, even promoters from heterologous organisms. Appropriate
promoters are known to the person skilled in the art and are
mentioned herein below. Further methods exists to modulate the
promoters of the genes of the invention, e.g. by modifying the
activity of transacting factors, meaning natural or artificial
transcription factors, which can bind to the promoter and influence
its activity. Furthermore it is possible to influence promoters of
interest by modifying upstream signaling components like receptors
or kinases, which are involved in the regulation of the promoter of
interest.
[0292] In a further embodiment, the process according to the
present invention comprises the following steps: [0293] (a)
selecting an organism or a part thereof expressing the polypeptide
of this invention; [0294] (b) mutagenizing the selected organism or
the part thereof; [0295] (c) comparing the activity or the
expression level of said polypeptide in the mutagenized organism or
the part thereof with the activity or the expression of said
polypeptide in the selected organisms or the part thereof; [0296]
(d) selecting the mutagenized organisms or parts thereof, which
comprise a decreased activity or expression level of said
polypeptide compared to the selected organism (a) or the part
thereof; [0297] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [0298] (f) harvesting the
organisms or parts therefore like e.g. seeds, fruits.
[0299] The organisms or part thereof show according to the herein
mentioned process of the invention an increased growth of the plant
compared to said control or selected plant or parts thereof
[0300] Advantageously the selected plants were mutagenized
according to the invention. According to the invention mutagenesis
is any change of the genetic information in the genome of a plant,
that means any structural or compositional change in the nucleic
acid preferably DNA of a plant that is not caused by normal
segregation or genetic recombiantion processes. Such mutations may
occur spontaneously, or may be induced by mutagens as described
below. Such change can be induced either randomly or selectively.
In both cases the genetic information of the plant is modified. In
general this leads to the situation that the activity of the gene
product of the relevant genes inside the cells or inside the plant
is reduced or repressed.
[0301] In case of the specific or site directed mutagenesis a
distinct gene is mutated and thereby its activity and/or the
activity or the encoded gene product is repressed, reduced,
decreased or deleted. In the event of a random mutagenesis one or
more genes are mutated by chance and their activities and/or the
activities of their gene products are repressed, reduced, decreased
or deleted, preferably decreased or deleted.
[0302] For the purpose of a mutagenesis of a huge population of
plants, such population can be transformed with a DNA population or
a DNA bank, which are useful for the inhibition of as much as
possible genes of a plant, preferably all genes. With this method
it is possible to statistically mutagenize nearly all genes of a
plant by the integration of an advantageously identified
DNA-fragment. Afterwards the skilled worker can easily identify the
knocked out event. For the mutagenesis of plants EMS, T-DNA and/or
transposon mutagenesis is preferred.
[0303] In the event of a random mutagenesis a huge number of plants
are treated with a mutagenic agent. The amount of said agent and
the intensity of the treatment will be chosen in such a manner that
statistically nearly every gene is mutated. The process for the
random mutagensis as well as the respective agents is well known by
the skilled person. Such methods are disclosed for example by A. M.
van Harten [(1998), "Mutation breeding: theory and practical
applications", Cambridge University Press, Cambridge, UK], E
Friedberg, G Walker, W Siede [(1995), "DNA Repair and Mutagenesis",
Blackwell Publishing], or K. Sankaranarayanan, J. M. Gentile, L. R.
Ferguson [(2000) "Protocols in Mutagenesis", Elsevier Health
Sciences]. As the skilled worker knows the spontaneous mutation
rate in the cells of an organism is very low and that a large
number of chemical, physical or biological agents are available for
the mutagenesis of organisms. These agents are named as mutagens or
mutagenic agents. As mentioned before three different kinds of
mutagens chemical, physical or biological agents are available.
[0304] There are different classes of chemical mutagens, which can
be separated by their mode of action. For example base analogues
such as 5-bromouracil, 2-amino purin. Other chemical mutagens are
interacting with the DNA such as sulphuric acid, nitrous acid,
hydroxylamine; or other alkylating agents such as monofunctional
agents like ethyl methane-sulfonate (=EMS), dimethylsulfate, methyl
methanesulfonate, bifunctional like dichloroethyl sulphide,
Mitomycin, Nitrosoguanidine-dialkylnitrosamine, N-Nitrosoguanidin
derivatives, N-alkyl-N-nitro-N-nitroso-guanidine, intercalating
dyes like Acridine, ethidium bromide.
[0305] Physical mutagens are for example ionizing irradiation
(X-ray), UV irradiation. Different forms of irradiation are
available and they are strong mutagens. Two main classes of
irradiation can be distinguished: a) non-ionizing irradiation such
as UV light or ionizing irradiation such as X-ray. Biological
mutagens are for example transposable elements for example IS
elements such as IS100, transposons such as Tn5, Tn10, Tn903, Tn916
or Tn1000 or phages like Mu.sup.amplac, P1, T5, .lamda.plac etc.
Methods for introducing this phage DNA into the appropriate
microorganism are well known to the skilled worker (see
Microbiology, Third Edition, Eds. Davis, B. D., Dulbecco, R.,
Eisen, H. N. and Ginsberg, H. S., Harper International Edition,
1980). The common procedure of a transposon mutagenesis is the
insertion of a transposable element within a gene or nearby for
example in the promotor or terminator region and thereby leading to
a loss of the gene function. Procedures to localize the transposon
within the genome of the organisms are well known by a person
skilled in the art. For transposon mutagenesis in plants the maize
transposon systems Activator-Dissociation (Ac/Ds) and
Enhancer-Supressor mutator (En/Spin) are known to the worker
skilled in the art but other transposon systems might be similarly
useful. The transposons can be brought into the plant genomes by
different available standard techniques for plant transformations.
Another type of biological mutagenesis in plants includes the T-DNA
mutagenesis, meaning the random integration of T-DNA sequences into
the plant genome [Feldmann, K. A. (1991) T-DNA insertion
mutagenesis in Arabidopsis: Mutational spectrum. Plant J. 1,
71-82]. The event in which the gene of interest is mutated can
later be searched by PCR- or other high throughput technologies
[Krysan et al., (1999) T_DNA as an insertional mutagen in
Arabidopsis, Plant Cell, 11, 2283-2290].
[0306] Another very efficient method is the introduction of
mutations into the genome of bacteria with the aid of transposons
(=Tn). Transposons have some common properties, which make them
useful as tool for the mutagenesis. Such properties are for example
ubiquitous finding in nature for example they are found on
chromosomes, plasmids and phages. The transposition of the
transposons in the genome is rec-independent and has a general
frequency of 10.sup.-4-10.sup.-7. Transposons are in the possession
of an encoded transposase and inverted terminal repeats at their
ends. Furthermore they need for integration in the genome a minimal
target sequence specificity, bordering on random. Like plasmids
they often confer antibiotic resistance. As an advantage they
generate polar mutations. Three kinds of transposons are
distinguished from one another a) conservative transposons: copy
number doesn't increase upon transpositon; b) replicative
transposons: transposon is copied upon transposition resulting in
two copies and c) conjugative transposons: transposon encodes Tra
functions, excises and transfers to another host. As the skilled
worker knows methods have been developed which facilitate the
introduction of transposable elements into a wide variety of both
grain negative and gram positive bacteria. Therefore transposable
elements can be introduced into the genome of nearly every
bacteria. They insert somewhat randomly thus causing insertion
mutations. Since the average bacterial species has approximately
3000 genes, one can saturate the chromosome with ease. Furthermore,
the transposon provides a molecular tag, which can be subsequently
used to identify the mutated gene and clone it. In combination with
genomics, transposons are a powerful approach to mutate distinct
genes. As mentioned before there are many different methods to
introduce transposons into bacteria. The choice will depend on the
nature of the target bacterium. Transposons can be introduced into
the genome of a bacteria for example with the aid of a
temperature-sensitive replicon, a so called bump plasmid by
introduction of an incompatible replicon, a transfer plasmid
lacking essential replication protein supplied in trans in donor
cell or phage that lacks replication in the host organism.
Typically well known transposons are Tn5, Tn10, Tn903, Tn916,
Tn1000 etc.
[0307] Other methods are for example the introduction of mutation
with the aid of viruses such as bacteriophages such as P1, P22, T2,
T3, T5, T7, Mu.sup.amplac, Mu, Mul, MuX, miniMu, .lamda.,
.lamda.plac or insertion elements such as IS3, IS100, IS900 etc.
Again the whole genome of the bacteria is randomly mutagenized.
Mutants can be easily identified.
[0308] Another method to disrupt the nucleic acid sequence of the
invention and thereby reducing, decreasing or deleting the
biological activity of the encoded polypeptide can be reached by
homologous recombination with an altered nucleic acid sequence of
the invention. The nucleic acid sequences of the invention can
therefore be altered by one or more point mutations, deletions, or
inversions, but still encodes a functional protein of the invention
or a non-functional protein. In another embodiment of the
invention, one or more of the regulatory regions (e.g., a promoter,
repressor, or inducer) of the gene encoding the protein of the
invention has been altered (e.g., by deletion, truncation,
inversion, or point mutation) such that the expression of the
corresponding gene is modulated that means reduced, decreased or
deleted.
[0309] Preferably a chemical or biochemical procedure is used for
the mutagenesis of the organisms. A preferred chemical method is
the mutagenesis with N-methyl-N-nitro-nitrosoguanidine.
[0310] Other biological methods are disclosed by Spee et al.
(Nucleic Acids Research, Vol. 21, No. 3, 1993: 777-778). Spee et
al. teaches a PCR method using dITP for the random mutagenesis.
This method described by Spee et al. was further improved by Rellos
et al. (Protein Expr. Purif., 5, 1994: 270-277). The use of an in
vitro recombination technique for molecular mutagenesis is
described by Stemmer (Proc. Natl. Acad. Sci. USA, Vol. 91, 1994:
10747-10751). Moore et al. (Nature Biotechnology Vol. 14, 1996:
458-467) describe the combination of the PCR and recombination
methods for increasing the enzymatic activity of an esterase toward
a para-nitrobenzyl ester. Another route to the mutagenesis of
enzymes is described by Greener et al. in Methods in Molecular
Biology (Vol. 57, 1996: 375-385). Greener et al. use the specific
Escherichia coli strain XL1-Red to generate Escherichia coli
mutants which have increased antibiotic resistance.
[0311] In one embodiment, the protein according to the invention or
the nucleic acid molecule characterized herein originates from an
eukaryotic or prokaryotic organism such as a non-human animal, a
plant, a microorganism such as a fungi, a yeast, an alga, a diatom
or a bacterium. Nucleic acids, which advantageously can be used in
the inventive process originate from plants, for example the family
Brassicaceae, in particular the genus Arabidopsis and the
especially advantageous from the species Arabidopsis thaliana. In
addition nucleic acid sequences, which advantageously can be used
in the inventive process originate from plants such as maize, soja,
canola, wheat, barley, triticale, rice, linseed, sunflower or
potato. The Accessions corresponding to the SEQ ID NO: 2 of the
polypeptide of this invention can be taken from the following table
for example. Sequence SEQ ID NO: 2 is deposited under the Genebank
Accession number At5g 64410.
TABLE-US-00003 SEQ ID NO: Accession Organism Protein at5g64410
Arabidopsis thaliana 2 PIR|D71430 Arabidopsis thaliana 4 PIR|F86233
Arabidopsis thaliana 6 PIR|S50773 Saccharomyces cerevisiae 8
PIR|S58824 Saccharomyces cerevisiae 10 PIR|T01900 Arabidopsis
thaliana 12 PIR|T05878 Arabidopsis thaliana 14 PIR|T08925
Arabidopsis thaliana 16 PIR|T38497 Schizosaccharomyces 18 pombe
PIR|T41655 Schizosaccharomyces 20 pombe SPTREMBL|O14411 Candida
albicans 22 SPTREMBL|Q6TUA0 Zea mays 24 SPTREMBL|Q6YUP9 Oryza
sativa 26 SPTREMBL|O6Z0P5 Oryza sativa 28 SPTREMBL|Q6Z8U6 Oryza
sativa 30 SPTREMBL|Q6ZCL0 Oryza sativa 32 SPTREMBL|Q750H8 Ashbya
gossypii 34 SPTREMBL|Q75BG1 Ashbya gossypii 36 SPTREMBL|Q75CX1
Ashbya gossypii 38 SPTREMBL|Q75D67 Ashbya gossypii 40
SPTREMBL|Q75LM0 Oryza sativa 42 SPTREMBL|Q7F9L4 Oryza sativa 44
SPTREMBL|Q7RW19 Neurospora crassa 46 SPTREMBL|Q7S189 Neurospora
crassa 48 SPTREMBL|Q7S4I2 Neurospora crassa 50 SPTREMBL|Q7S4Q8
Neurospora crassa 52 SPTREMBL|Q7S882 Neurospora crassa 54
SPTREMBL|Q7S9V4 Neurospora crassa 56 SPTREMBL|Q7SC23 Neurospora
crassa 58 SPTREMBL|Q7SEW1 Neurospora crassa 60 SPTREMBL|Q8H641
Oryza sativa 62 SPTREMBL|Q8S2I4 Oryza sativa 64 SPTREMBL|Q8WZL3
Yarrowia lipolytica 66 SPTREMBL|Q93ZI8 Arabidopsis thaliana 68
SPTREMBL|Q940I5 Arabidopsis thaliana 70 SPTREMBL|Q9FG72 Arabidopsis
thaliana 72 SPTREMBL|FQ9FJD1 Arabidopsis thaliana 74
SPTREMBL|Q9FJD2 Arabidopsis thaliana 76 SPTREMBL|Q9LWV0 Oryza
sativa 78 SPTREMBL|Q9LWW1 Oryza sativa 80 SPTREMBL|Q9LWW2 Oryza
sativa 82 SPTREMBL|Q9P939 Schizophyllum commune 84
SWISSPROT|ISP4_SCHPO Schizosaccharomyces 86 pombe
[0312] Accordingly, in one embodiment, the invention relates to an
isolated nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of: [0313] a) nucleic
acid molecule which encodes a polypeptide comprising the
polypeptide shown in, SEQ ID NO: 2 [0314] b) nucleic acid molecule
comprising the polynucleotide shown in SEQ ID NO: 1; [0315] nucleic
acid molecule comprising a nucleic acid sequence, which, as a
result of the degeneracy of the genetic code, can be derived from a
polypeptide sequence depicted (b) and having the biological
activity represented by the protein as depicted in SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ
ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:
48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ
ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108,
SEQ ID NO: 110, SEQ ID NO: 112; [0316] c) nucleic acid molecule
encoding a polypeptide having at least 50% identity with the amino
acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) or (c) and having the biological activity
represented by the protein as depicted in SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112; [0317] d) nucleic acid molecule which
comprises a polynucleotide, which is obtained by amplifying a cDNA
library or a genomic library using the primers in SEQ ID NO: 92 and
SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID
NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO:
101 and SEQ ID NO: 102, [0318] e) nucleic acid molecule encoding a
polypeptide, which is isolated with the aid of monoclonal and/or
polyclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (c) and having the biological
activity represented by the protein as depicted in SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ
ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:
48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ
ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108,
SEQ ID NO: 110, SEQ ID NO: 112; [0319] f) nucleic acid molecule
encoding a polypeptide comprising the consensus sequence shown in
SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID
NO: 91, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267 or SEQ ID
NO: 268 and having the biological activity represented by the
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112; [0320] g) nucleic acid molecule encoding a polypeptide having
the biological activity represented by the protein as depicted in
SEQ ID NO: 2; [0321] h) nucleic acid molecule which is obtainable
by screening a suitable library under stringent hybridisation
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (c) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (i) and encoding
a polypeptide having the biological activity represented by the
protein as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112; [0322] i) or which comprises a sequence which is complementary
thereto; whereby the nucleic acid molecule according to (a) to (j)
is at least in one or more nucleotides different from the sequence
depicted in SEQ ID NO: 1 and/or which encodes a protein which
differs at least in one, two, three, four, five, six, seven, eight,
nine, ten or more amino acids from the protein sequences depicted
in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,
SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID
NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,
SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID
NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,
SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID
NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,
SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID
NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO:
106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112.
[0323] In a further embodiment, the nucleic acid molecule of the
present invention is at least 30% identical to the nucleic acid
sequence depicted in SEQ ID NO: 1 and less than 100%, preferably
less than 99.999%, 99.99% or 99.9%, more preferably less than 99%,
985, 97%, 96% or 95% identical to the sequence shown in SEQ ID NO:
1.
[0324] Preferably, the nucleic acid molecule also does not encode a
polypeptide as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID
NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID
NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112.
[0325] In another embodiment, the nucleic acid molecule depicted in
SEQ ID NO: 1 does not encode a protein of the sequence shown in,
SEQ ID NO: 2. That means the protein sequences depicted in SEQ ID
NO: 2 does not consist of the sequence shown in SEQ ID NO: 2. In a
further embodiment, the protein of the present invention is at
least 30% identical to protein sequence depicted in SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ
ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:
48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ
ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108,
SEQ ID NO: 110, SEQ ID NO: 112 and less than 100%, preferably less
than 99.999%, 99.99% or 99.9%, more preferably less than 99%, 985,
97%, 96% or 95% identical to the sequence shown in SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ
ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:
48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ
ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108,
SEQ ID NO: 110, SEQ ID NO: 112.
[0326] The nucleic acid sequences used in the process are
advantageously introduced in a nucleic acid construct, preferably
an expression cassette, which allows the reduction, depression etc.
of the nucleic acid molecules in a plant.
[0327] Accordingly, the invention also relates to a nucleic acid
construct, preferably to an expression construct, comprising the
nucleic acid molecule of the present invention or a fragment
thereof functionally linked to one or more regulatory elements or
signals.
[0328] As described herein, the nucleic acid construct can also
comprise further genes, which are to be introduced into the plant
or plant cells. It is possible and advantageous to introduce into,
and express in, the host plant regulatory genes such as genes for
inductors, repressors or enzymes, which, owing to their enzymatic
activity, engage in the regulation of one or more genes of a
biosynthetic pathway or in regulation of gene expression or
modification of metabolism. These genes can be of heterologous or
homologous origin. Moreover, further biosynthesis genes may
advantageously be present, or else these genes may be located on
one or more further nucleic acid constructs. Genes, which are
advantageously additionally employed as plant growth increasing
genes are transcription factors, general signaling components like
kinases and phosphateses, cell cycle and cell cycle related genes,
genes involved in the production and reception of phytohormes and
genes of the amino acid metabolism, of glycolysis, of the
tricarboxylic acid metabolism or their combinations.
[0329] As described herein, regulator sequences or factors can have
a positive effect on preferably the gene expression of the genes
introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing RNA
stability. On the other hand the nucleic acid molecules of the
invention and the gene products are reduced, decreased or deleted
to increase the plant growth as described by the invention.
[0330] In principle, the nucleic acid construct can comprise the
herein described regulator sequences and further sequences relevant
for the reduction of the expression of nucleic acid molecules of
the invention and on the other side for the expression of
additional genes in the construct. Thus, the nucleic acid construct
of the invention can be used as expression cassette and thus can be
used directly for introduction into the plant, or else they may be
introduced into a vector. Accordingly in one embodiment the nucleic
acid construct is an expression cassette comprising a microorganism
promoter or a microorganism terminator or both. In another
embodiment the expression cassette encompasses a plant promoter or
a plant terminator or both.
[0331] Accordingly, in one embodiment, the process according to the
invention comprises the following steps: [0332] 1. introduction of
a nucleic acid construct comprising the nucleic acid molecule of
the invention, which encodes the polypeptide of the present
invention; or [0333] 2. introduction of a nucleic acid molecule,
including regulatory sequences or factors, which expression
decreases the expression of the polypeptide of the invention; in a
plant cell, a plant tissue, a plant or a plant part thereof, and
[0334] 3. repressing the polypeptide encoded by the nucleic acids
of the invention by the nucleic acid construct or the nucleic acid
molecule mentioned under (a) or (b) in the cell or the plant.
[0335] After the introduction and expression of the nucleic acid
construct the trans-genic plant, preferably a crop plant, or a part
thereof, or plant cell or tissue is advantageously cultured and
subsequently harvested.
[0336] To introduce a nucleic acid molecule for example an RNAi,
antisense nucleic acid sequence or a mutagenized nucleic acid
sequence into a nucleic acid construct, e.g. as part of an
expression cassette, which leads to a reduced activity and/or
expression of the respective gene, the codogenic gene segment or a
part of it or the untranslated regions are as advantageously
subjected to an amplification and ligation reaction in the manner
known by a skilled person. It is preferred to follow a procedure
similar to the protocol for the Pfu DNA polymerase or a Pfu/Taq DNA
polymerase mixture. The primers are selected according to the
sequence to be amplified. Additional possibilities include the 5'
or 3' untranslated regions or the promoter region. After the
amplification, the amplificate is expediently analyzed. For
example, the analysis may consider quality and quantity and be
carried out following separation by gel electrophoresis.
Thereafter, the amplificate can be purified following a standard
protocol (for example Qiagen). An aliquot of the purified
amplificate is then available for the subsequent cloning step.
Suitable cloning vectors are generally known to the skilled worker
[Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New
York-Oxford, 1985, SBN 0 444 904018)].
[0337] They include, in particular, vectors which are capable of
replication in easy to handle cloning systems like bacterial yeast
or insect cell based (e.g. baculovirus expression) systems, that is
to say especially vectors which ensure efficient cloning in E.
coli, and which make possible the stable transformation of plants.
Vectors, which must be mentioned, in particular are various binary
and cointegrated vector systems, which are suitable for the
T-DNA-mediated transformation. Such vector systems are generally
characterized in that they contain at least the vir genes, which
are required for the Agrobacterium-mediated transformation, and the
T-DNA border sequences.
[0338] In general, vector systems preferably also comprise further
cis-regulatory regions such as promoters and terminators and/or
selection markers by means of which suitably transformed organisms
can be identified. While vir genes and T-DNA sequences are located
on the same vector in the case of cointegrated vector systems,
binary systems are based on at least two vectors, one of which
bears vir genes, but no T-DNA, while a second one bears T-DNA, but
no vir gene. Owing to this fact, the last-mentioned vectors are
relatively small, easy to manipulate and capable of replication in
E. coli and in Agrobacterium. These binary vectors include vectors
from the series pBIB-HYG, pPZP, pBecks, pGreen. Those, which are
preferably used in accordance with the invention, are Bin19,
pBI101, pBinAR, pSun, pGPTV and pCAMBIA or pHELLESGATE. An overview
of binary vectors and their use is given by Hellens et al, Trends
in Plant Science (2000) 5, 446-451.
[0339] For a vector preparation, vectors may first be linearized
using restriction endonuclease(s) and then be modified
enzymatically in a suitable manner. Thereafter, the vector is
purified, and an aliquot is employed in the cloning step. In the
cloning step, the enzyme-cleaved and, if required, purified
amplificate is cloned together with similarly prepared vector
fragments, using ligase. In this context, a specific nucleic acid
construct, or vector or plasmid construct, may have one or else
more codogenic or non codogenic gene segments. The gene segments in
these constructs are preferably linked operably to regulatory
sequences. The regulatory sequences include, in particular, plant
sequences like the above-described promoters and terminators. The
constructs can advantageously be propagated stably in
microorganisms, in particular Escherichia coli and/or Agrobacterium
tumefaciens, under selective conditions and enable the transfer of
heterologous DNA into plants or other microorganisms. In accordance
with a particular embodiment, the constructs are based on binary
vectors (overview of a binary vector: Hellens et al., 2000). As a
rule, they contain prokaryotic regulatory sequences, such as
replication origin and selection markers, for the multiplication in
microorganisms such as Escherichia coli and Agrobacterium
tumefaciens. Vectors can further contain agrobacterial T-DNA
sequences for the transfer of DNA into plant genomes or other
eukaryotic regulatory sequences for transfer into other eukaryotic
cells, e.g. Saccharomyces sp. For the transformation of plants, at
least the right border sequence, which comprises approximately 25
base pairs, of the total agrobacterial T-DNA sequence is required.
Usually, the plant transformation vector constructs according to
the invention contain T-DNA sequences both from the right and from
the left border region, which contain expedient recognition sites
for site-specific acting enzymes, which, in turn, are encoded by
some of the vir genes.
[0340] Advantageously preferred in accordance with the invention
are host organisms of the genus Agrobacterium tumefaciens or
plants. Preferred plants are selected from among the families
Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Apiaceae,
Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cactaceae,
Caricaceae, Caryophyllaceae, Cannabaceae, Convolvulaceae,
Chenopodiaceae, Elaeagnaceae, Geraniaceae, Gramineae, Juglandaceae,
Lauraceae, Leguminosae, Linaceae, Cucurbitaceae, Cyperaceae,
Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae, Papaveraceae,
Rosaceae, Salicaceae, Solanaceae, Arecaceae, Iridaceae, Liliaceae,
Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae, Ranunculaceae,
Carifolaceae, Rubiaceae, Scrophulariaceae, Ericaceae, Polygonaceae,
Violaceae, Juncaceae, Poaceae, perennial grass, fodder crops,
vegetables and ornamentals.
[0341] Especially preferred are plants selected from the groups of
the families Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae,
Fabaceae, Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae.
Especially advantageous are, in particular, crop plants.
Accordingly, an advantageous plant preferably belongs to the group
of the genus peanut, oilseed rape, canola, sunflower, safflower,
olive, sesame, hazelnut, almond, avocado, bay, pumpkin/squash,
linseed, soya, pistachio, borage, maize, wheat, rye, oats, sorghum
and millet, triticale, rice, barley, cassava, potato, sugarbeet,
fodder beet, egg plant, and perennial grasses and forage plants,
oil palm, vegetables (brassicas, root vegetables, tuber vegetables,
pod vegetables, fruiting vegetables, onion vegetables, leafy
vegetables and stem vegetables), buckwheat, Jerusalem artichoke,
broad bean, vetches, lentil, alfalfa, dwarf bean, lupin, clover and
lucerne. Further preferred plants are mentioned above.
[0342] In order to introduce, into a plant, the nucleic acid
molecule of the invention or used in the process according to the
invention for example an RNAi, antisense nucleic acid sequence or a
mutagenized nucleic acid sequence, it has proved advantageous first
to transfer them into an intermediate host, for example a bacterium
or a eukaryotic unicellular cell. The transformation into E. coli,
which can be carried out in a manner known per se, for example by
means of heat shock or electroporation, has proved itself expedient
in this context. Thus, the transformed E. coli colonies can be
analysed for their cloning efficiency. This can be carried out with
the aid of a PCR. Here, not only the identity, but also the
integrity, of the plasmid construct can be verified with the aid of
a defined colony number by subjecting an aliquot of the colonies to
said PCR. The amplificates are separated by electrophoresis and
assessed with regard to quantity and quality.
[0343] The nucleic acid constructs, which are optionally verified,
are subsequently used for the transformation of the plants or other
hosts, e.g. other eukaryotic cells or other prokaryotic cells. To
this end, it may first be necessary to obtain the constructs from
the intermediate host. For example, the constructs may be obtained
as plasmids from bacterial hosts by a method similar to
conventional plasmid isolation.
[0344] Gene silencing in plants can advantageously achieved by
transient transformation technologies, meaning that the nucleic
acids are preferably not integrated into the plant genome. Suitable
systems for transient plant transformations are for example
agrobacterium based and plant virus based systems. Details about
virus based transient systems and their use for gene silencing in
plants have been described in Lu et al. in Methods 2003, 30(4)
296-303. The use of agrobacterium for the transient expression of
nucleic acids in plants have been described for example by Fuentes
et al., 2003 in Biotechnol Appl Biochem. 2003 Nov. 21 online:
doi:10.1042/BA20030192.
[0345] A large number of methods for the transformation of plants
are known. Since, in accordance with the invention, a stable
integration of heterologous DNA into the genome of plants is
advantageous, the T-DNA-mediated transformation has proved
expedient in particular. For this purpose, it is first necessary to
transform suitable vehicles, in particular agrobacteria, with a
gene segment or the corresponding plasmid construct comprising the
nucleic acid molecule of the invention. This can be carried out in
a manner known per se. For example, said nucleic acid construct of
the invention, or said expression, antisense or RNAi construct or
said plasmid construct, which has been generated in accordance with
what has been detailed above, can be transformed into competent
agrobacteria by means of electroporation or heat shock. In
principle, one must differentiate between the formation of
cointegrated vectors on the one hand and the transformation with
binary vectors on the other hand. In the case of the first
alternative, the constructs, which comprise the codogenic gene
segment or the nucleic acid molecule of the invention have no T-DNA
sequences, but the formation of the cointegrated vectors or
constructs takes place in the agrobacteria by homologous
recombination of the construct with T-DNA. The T-DNA is present in
the agrobacteria in the form of Ti or Ri plasmids in which
exogenous DNA has expediently replaced the oncogenes. If binary
vectors are used, they can be transferred to agrobacteria either by
bacterial conjugation or by direct transfer. These agrobacteria
expediently already comprise the vector bearing the vir genes
(currently referred to as helper Ti(Ri) plasmid).
[0346] In addition the stable transformation of plastids is of
advantageous because plastids are inherited maternally in most
crops reducing or eliminating the risk of transgene flow through
pollen. The process of the transformation of the chloroplast genome
is generally achieved by a process which has been schematically
displayed in Klaus et al., 2004, Nature Biotechnology 22(2),
225-229).
[0347] Briefly the sequences to be transformed are cloned together
with a selectable marker gene between flanking sequences homologous
to the chloroplast genome. These homologous flanking sequences
direct site specific intergration into the plastome. Plastidal
transformation has been described for many different plant species
and an overview can be taken from Bock et al. [(2001) Transgenic
plastids in basic research and plant biotechnology. J Mol. Biol.
2001 Sep. 21; 312(3):425-38] or Maliga, P [Progress towards
commercialization of plastid transformation technology. Trends
Biotechnol. 21, 20-28 (2003)]. Further biotechnological progress
has recently been reported in form of marker free plastid
transformants, which can be produced by a transient cointegrated
maker gene [Klaus et al., 2004, Nature Biotechnology 22(2),
225-229].
[0348] One or more markers may expediently also be used together
with the nucleic acid construct, or the vector of the invention
and, if plants or plant cells shall be transformed together with
the T-DNA, with the aid of which the isolation or selection of
transformed organisms, such as agrobacteria or transformed plant
cells, is possible. These marker genes enable the identification of
a successful transfer of the nucleic acid molecules according to
the invention via a series of different principles, for example via
visual identification with the aid of fluorescence, luminescence or
in the wavelength range of light which is discernible for the human
eye, by a resistance to herbicides or antibiotics, via what are
known as nutritive markers (auxotrophism markers) or antinutritive
markers, via enzyme assays or via phytohormones. Examples of such
markers which may be mentioned are GFP (=green fluorescent
protein); the luciferin/luceferase system, the
(.beta.-galactosidase with its colored substrates, for example
X-Gal, the herbicide resistances to, for example, imidazolinone,
glyphosate, phosphinothricin or sulfonylurea, the antibiotic
resistances to, for example, bleomycin, hygromycin, streptomycin,
kanamycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,
geneticin (G418), spectinomycin or blasticidin, to mention only a
few, nutritive markers such as the utilization of mannose or
xylose, or antinutritive markers such as the resistance to
2-deoxyglucose. This list is a small number of possible markers.
The skilled worker is very familiar with such markers. Different
markers are preferred, depending on the organism and the selection
method.
[0349] As a rule, it is desired that the plant nucleic acid
constructs be flanked by T-DNA at one or both sides of the gene
segment. This is particularly useful when bacteria of the species
Agrobacterium tumefaciens or Agrobacterium rhizogenes are used for
the transformation. A method, which is preferred in accordance with
the invention, is the transformation with the aid of Agrobacterium
tumefaciens. However, biolistic methods may also be used
advantageously for introducing the sequences in the process
according to the invention, and the introduction by means of PEG is
also possible. The transformed agrobacteria can be grown in the
manner known per se and are thus available for the expedient
transformation of the plants. The plants or plant parts to be
transformed are grown or provided in the customary manner. The
transformed agrobacteria are subsequently allowed to act on the
plants or plant parts until a sufficient transformation rate is
reached. Allowing the agrobacteria to act on the plants or plant
parts can take different forms. For example, a culture of
morphogenic plant cells or tissue may be used. After the T-DNA
transfer, the bacteria are, as a rule, eliminated by antibiotics,
and the regeneration of plant tissue is induced. This is done in
particular using suitable plant hormones in order to initially
induce callus formation and then to promote shoot development.
[0350] The transfer of nucleic acid constructs into the genome of a
plant is called transformation. In doing this the methods described
for the transformation and regeneration of plants from plant
tissues or plant cells are utilized for transient or stable
transformation. An advantageous transformation method is the
transformation in planta. To this end, it is possible, for example,
to allow the agrobacteria to act on plant seeds or to inoculate the
plant meristem with agrobacteria. It has proved particularly
expedient in accordance with the invention to allow a suspension of
transformed agrobacteria to act on the intact plant or at least the
flower primordia. The plant is subsequently grown on until the
seeds of the treated plant are obtained (Clough and Bent, Plant J.
(1998) 16, 735-743). To select transformed plants, the plant
material obtained in the transformation is, as a rule, subjected to
selective conditions so that transformed plants can be
distinguished from untransformed plants. For example, the seeds
obtained in the above-described manner can be planted and, after an
initial growing period, subjected to a suitable selection by
spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into
plants. Further advantageous transformation methods, in particular
for plants, are known to the skilled worker and are described
hereinbelow.
[0351] A further advantageously suitable methods are protoplast
transformation by poly(ethylene glycol)-induced DNA uptake, the
"biolistic" method using the gene cannon--referred to as the
particle bombardment method, electroporation, the incubation of dry
embryos in DNA solution, microinjection and gene transfer mediated
by Agrobacterium. Said methods are described by way of example in
B. Jenes et al., Techniques for Gene Transfer, in: Transgenic
Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R.
Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant
Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids
or the construct to be expressed is preferably cloned into a
vector, which is suitable for transforming Agrobacterium
tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12
(1984) 8711). Agrobacteria transformed by such a vector can then be
used in known manner for the transformation of plants, in
particular of crop plants such as by way of example tobacco plants,
for example by bathing bruised leaves or chopped leaves in an
agrobacterial solution and then culturing them in suitable media.
The transformation of plants by means of Agrobacterium tumefaciens
is described, for example, by Hofgen and Willmitzer in Nucl. Acid
Res. (1988) 16, 9877 or is known inter alia from F. F. White,
Vectors for Gene Transfer in Higher Plants; in Transgenic Plants,
Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu,
Academic Press, 1993, pp. 15-38.
[0352] The abovementioned nucleic acid molecules can be cloned into
the nucleic acid constructs or vectors according to the invention
in combination together with further genes, or else different genes
are introduced by transforming several nucleic acid constructs or
vectors (including plasmids) into a host cell, advantageously into
a plant cell or a microorgansims.
[0353] In addition to the sequences mentioned in SEQ ID NO: 1 and
SEQ ID NO: 113 or its derivatives, it is advantageous additionally
to express and/or mutate further genes in the organisms. It is also
possible that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased growth desired since, for
example, feedback regulations no longer exist to the same extent or
not at all. In addition it might be advantageously to combine the
sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID
NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,
SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,
SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID
NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID
NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,
SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID
NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111 or SEQ ID
NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287 which are
reduced in the biological activity, with genes which generally
support or enhance the growth or yield of the target organismen,
for example genes which lead to faster a growth rate or genes which
for example produces stress-, pathogen, or herbicide resistant
plants.
[0354] In a further embodiment of the process of the invention,
therefore, organisms are grown, in which there is simultaneous
overexpression of at least one nucleic acid or one of the genes
which code for proteins selected from the group of gene products
consisting of aspartate kinase (lysC), of aspartate-semialdehyde
dehydrogenase (asd), of glyceraldehyde-3-phosphate dehydrogenase
(gap), of 3-phosphoglycerate kinase (pgk), of pyruvate carboxylase
(pyc), of triosephosphate isomerase (tpi), of homoserine
O-acetyltransferase (metA), of cystathionine y-synthase (metB), of
cystathionine gamma-lyase (metC), cystathionine .beta.-lyase, of
methionine synthase (metH), of serine hydroxymethyltransferase
(glyA), of O-acetylhomoserine sulfhydrylase (metY), of
methylenetetrahydrofolate reductase (metF), of phosphoserine
aminotransferase (serC), of phosphoserine phosphatase (serB), of
serine acetyltransferase (cysE), of cysteine synthase (cysK), of
homoserine dehydrogenase (horn) and S-adenosylmethionine synthase
(metX).
[0355] A further advantageous nucleic acid sequence, which can be
expressed in combination with the sequences used in the process
and/or the abovementioned biosynthesis genes is the sequence of the
ATP/ADP translocator as described in WO 01/20009. This ATP/ADP
translocator leads to an increased synthesis of the essential amino
acids lysine and/or methionine.
[0356] In a further advantageous embodiment of the process of the
invention, the organisms used in the process are those in which
simultaneously at least one of the aforementioned genes or one of
the aforementioned nucleic acids is mutated so that the activity of
the corresponding proteins is influenced by metabolites to a
smaller extent compared with the unmutated proteins, or not at all,
and that in particular the transport of oligopeptides according to
the invention is not impaired, or so that their specific enzymatic
activity is increased. This increased activity of the genes, which
are expressed in addition to the reduced, decreased or deleted
activities of the nucleic acid sequences of the invention, leads to
an increased plant growth. Less influence means in this connection
that the regulation of the enzymic activity is less by at least
10%, advantageously at least 20, 30 or 40%, particularly
advantageously by at least 50, 60 or 70%, compared with the
starting organism, and thus the activity of the enzyme is increased
by these figures mentioned compared with the starting organism. An
increase in the enzymatic activity means an enzymatic activity
which is increased by at least 10%, advantageously at least 20, 30
or 40%, particularly advantageously by at least 50, 60 or 70%,
compared with the starting organism. This leads to an increased
plant growth.
[0357] In a further advantageous embodiment of the process of the
invention, the organisms used in the process are those in which
simultaneously at least one of the genes selected from homoserine
kinase (thrB), threonine dehydratase (ilvA), threonine synthase
(thrC), meso-diaminopimelate D-dehydrogenase (ddh),
phosphoenolpyruvate carboxykinase (pck), glucose-6-phosphate
6-isomerase (pgi), pyruvate oxidase (poxB), dihydrodipicolinate
synthase (dapA), dihydrodipicolinate reductase (dapB) and
diaminopicolinate decarboxylase (lysA) or a threonin degrading
protein is attenuated, in particular by reducing the rate of
expression of the corresponding gene.
[0358] In another embodiment of the process of the invention, the
organisms used in the process are those in which simultaneously at
least one of the aforementioned nucleic acids or of the
aforementioned genes is mutated in such a way that the enzymatic
activity of the corresponding protein is partially reduced or
completely blocked. A reduction in the enzymatic activity means an
enzymatic activity, which is reduced by at least 10%,
advantageously at least 20, 30 or 40%, particularly advantageously
by at least 50, 60 or 70%, preferably more, compared with the
starting organism.
[0359] If it is intended to transform the host cell, in particular
the plant cell, with several constructs or vectors, the marker of a
preceding transformation must be removed or a further marker
employed in a following transformation. The markers can be removed
from the host cell, in particular the plant cell, as described
hereinbelow via methods with which the skilled worker is familiar.
In particular plants without a marker, in particular without
resistance to antibiotics, are an especially preferred embodiment
of the present invention.
[0360] In the process according to the invention, the nucleic acid
sequences used in the process according to the invention are
advantageously linked operably to one or more regulatory signals in
order to increase gene expression for example if RNAi or antisense
is used. These regulatory sequences are intended to enable the
specific expression of the genes or gene fragments. Depending on
the host organism for example plant or microorganism, this may
mean, for example, that the gene or gene fragment is expressed
and/or overexpressed after induction only, or that it is expressed
and/or overexpressed constitutive. These regulatory sequences are,
for example, sequences to which the inductors or repressors bind
and which thus regulate the expression of the nucleic acid. In
addition to these novel regulatory sequences, or instead of these
sequences, the natural regulation of these sequences may still be
present before the actual structural genes and, if appropriate, may
have been genetically modified so that the natural regulation has
been switched off and gene expression has been increased. However,
the nucleic acid construct of the invention suitable as expression
cassette (=expression construct=gene construct) can also be simpler
in construction, that is to say no additional regulatory signals
have been inserted before the nucleic acid sequence or its
derivatives, and the natural promoter together with its regulation
has not been removed. Instead, the natural regulatory sequence has
been mutated in such a way that regulation no longer takes place
and/or gene expression is increased. These modified promoters can
also be introduced on their own before the natural gene in the form
of part sequences (=promoter with parts of the nucleic acid
sequences according to the invention) in order to increase the
activity. Moreover, the gene construct can advantageously also
comprise one or more of what are known as enhancer sequences in
operable linkage with the promoter, and these enable an increased
expression of the nucleic acid sequence. Also, it is possible to
insert additional advantageous sequences at the 3' end of the DNA
sequences, such as, for example, further regulatory elements or
terminators.
[0361] The nucleic acid molecules, which encode proteins according
to the invention and nucleic acid molecules, which encode other
polypeptides may be present in one nucleic acid construct or vector
or in several ones. Advantageously, only one copy of the nucleic
acid molecule of the invention or its encoding genes is present in
the nucleic acid construct or vector. Several vectors or nucleic
acid construct or vector can be expressed together in the host
organism. The nucleic acid molecule or the nucleic acid construct
or vector according to the invention can be inserted in a vector
and be present in the cell in a free form. If a stable
transformation is preferred, a vector is used, which is stably
duplicated over several generations or which is else be inserted
into the genome. In the case of plants, integration into the
plastid genome or, in particular, into the nuclear genome may have
taken place. For the insertion of more than one gene in the host
genome the genes to be expressed are present together in one gene
construct, for example in above-described vectors bearing a
plurality of genes.
[0362] As a rule, regulatory sequences for the expression rate of a
gene are located upstream (5'), within, and/or downstream (3')
relative to the coding sequence of the nucleic acid molecule of the
invention or another codogenic gene segment. They control in
particular transcription and/or translation and/or the transcript
stability. The expression level is dependent on the conjunction of
further cellular regulatory systems, such as the protein
biosynthesis and degradation systems of the cell.
[0363] Regulatory sequences include transcription and translation
regulating sequences or signals, e.g. sequences located upstream
(5'), which concern in particular the regulation of transcription
or translation initiation, such as promoters or start codons, and
sequences located downstream (3'), which concern in particular the
regulation of transcription or translation termination and
transcript stability, such as polyadenylation signals or stop
codons. Regulatory sequences can also be present in transcribed
coding regions as well in transcribed non-coding regions, e.g. in
introns, as for example splicing sites.
[0364] Promoters for the regulation of expression of the nucleic
acid molecule according to the invention in a cell and which can be
employed are, in principle, all those which are capable of reducing
the transcription of the nucleic acid molecules or stimulating the
transcription of additional genes in the organisms in question,
such as microorganisms or plants, depending on the goal, which
should be reached by using said promotors. Suitable promoters,
which are functional in these organisms, are generally known. They
may take the form of constitutive or inducible promoters. Suitable
promoters can enable the development- and/or tissue-specific
expression in multi-celled eukaryotes; thus, leaf-, root-, flower-,
seed-, stomata-, tuber- or fruit-specific promoters may
advantageously be used in plants.
[0365] Promoters, which are particularly advantageous, are
constitutive, tissue or compartment specific and inducible
promoters. In general, "promoter" is understood as meaning, in the
present context, a regulatory sequence in a nucleic acid molecule,
which mediates the expression of a coding sequence segment of a
nucleic acid molecule. In general, the promoter is located upstream
to the coding sequence segment. Some elements, for example
expression-enhancing elements such as enhancer may, however, also
be located down-stream or even in the transcribed region.
[0366] In principle, it is possible to use natural promoters
together with their regulatory sequences, such as those mentioned
above, for the novel process. It is also possible advantageously to
use synthetic promoters, either additionally or alone, in
particular when they mediate seed-specific expression such as
described in, for example, WO 99/16890.
[0367] The expression of the nucleic acid molecules used in the
process may be desired alone or in combination with other genes or
nucleic acids. Multiple nucleic acid molecules conferring
repression or expression of advantageous genes, depending on the
goal to be reached, can be introduced via the simultaneous
transformation of several individual suitable nucleic acid
constructs, i.e. expression constructs, or, preferably, by
combining several expression cassettes on one construct. It is also
possible to transform several vectors with in each case several
expression cassettes stepwise into the recipient organismen.
[0368] As described above, the transcription of the genes, which
are in addition to the genes of the invention introduced should
advantageously be terminated by suitable terminators at the 3' end
of the biosynthesis genes introduced (behind the stop codon). A
terminator, which may be used for this purpose is, for example, the
OCS1 terminator, the nos3 terminator or the 35S terminator. As is
the case with the promoters, different terminator sequences should
be used for each gene.
[0369] Different plant promoters such as, for example, the USP, the
LegB4-, the DC3 promoter or the ubiquitin promoter from parsley or
other herein mentioned promoter and different terminators may
advantageously be used in the nucleic acid construct. Further
useful plant promoters are for example the maize ubiquitin
promoter, the ScBV (Sugarcaine bacilliform virus) promoter, the
lpt2 or lpt1-gene promoters from barley (WO 95/15389 and WO
95/23230) or those described in WO 99/16890 (promoters from the
barley hordein-gene, the rice glutelin gene, the rice oryzin gene,
the rice prolamin gene, the wheat gliadin gene, wheat glutelin
gene, the maize zein gene, the oat glutelin gene, the Sorghum
kasirin-gene, the rye secalin gene).
[0370] In order to ensure the stable integration, into the
transgenic plant, of nucleic acid molecules used in the process
according to the invention in combination with further biosynthesis
genes over a plurality of generations, each of the coding regions
used in the process should be expressed under the control of its
own, preferably unique, promoter since repeating sequence motifs
may lead to recombination events or to silencing or, in plants, to
instability of the T-DNA.
[0371] The nucleic acid construct is advantageously constructed in
such a way that a promoter is followed by a suitable cleavage site
for insertion of the nucleic acid to be expressed, advantageously
in a polylinker, followed, if appropriate, by a terminator located
behind the polylinker. If appropriate, this order is repeated
several times so that several nucleic acid sequences are combined
in one construct and thus can be introduced into the transgenic
plant in order to be expressed. The sequence is advantageously
repeated up to three times. For the expression, the nucleic acid
sequences are inserted via the suitable cleavage site, for example
in the polylinker behind the promoter. It is advantageous for each
nucleic acid sequence to have its own promoter and, if appropriate,
its own terminator, as mentioned above. However, it is also
possible to insert several nucleic acid sequences behind a promoter
and, if appropriate, before a terminator if a polycistronic
transcription is possible in the host or target cells. In this
context, the insertion site, or the sequence of the nucleic acid
molecules inserted, in the nucleic acid construct is not decisive,
that is to say a nucleic acid molecule can be inserted in the first
or last position in the cassette without this having a substantial
effect on the expression. However, it is also possible to use only
one promoter type in the construct. However, this may lead to
undesired recombination events or silencing effects, as said.
[0372] Accordingly, in a preferred embodiment, the nucleic acid
construct according to the invention confers expression of the
nucleic acid molecule of the invention, and, optionally further
genes, in a plant and comprises one or more plant regulatory
elements. Said nucleic acid construct according to the invention
advantageously encompasses a plant promoter or a plant terminator
or a plant promoter and a plant terminator.
[0373] A "plant" promoter comprises regulatory elements, which
mediate the expression of a coding sequence segment in plant cells.
Accordingly, a plant promoter need not be of plant origin, but may
originate from viruses or microorganisms, in particular for example
from viruses which attack plant cells.
[0374] The plant promoter can also originate from a plant cell,
e.g. from the plant, which is transformed with the nucleic acid
construct or vector as described herein. This also applies to other
"plant" regulatory signals, for example in "plant" terminators.
[0375] A nucleic acid construct suitable for plant expression
preferably comprises regulatory elements which are capable of
controlling the expression of genes in plant cells and which are
operably linked so that each sequence can fulfill its function.
Accordingly, the nucleic acid construct can also comprise
transcription terminators. Examples for transcriptional termination
are polyadenylation signals. Preferred polyadenylation signals are
those which originate from Agrobacterium tumefaciens T-DNA, such as
the gene 3 of the Ti plasmid pTiACH5, which is known as octopine
synthase (Gielen et al., EMBO J. 3 (1984) 835 et seq.) or
functional equivalents thereof, but all the other terminators which
are functionally active in plants are also suitable.
[0376] The nucleic acid construct suitable for plant expression
preferably also comprises other operably linked regulatory elements
such as translation enhancers, for example the overdrive sequence,
which comprises the tobacco mosaic virus 5'-untranslated leader
sequence, which increases the protein/RNA ratio (Gallie et al.,
1987, Nucl. Acids Research 15:8693-8711).
[0377] For expression in plants, the nucleic acid molecule must, as
described above, be linked operably to or comprise a suitable
promotor which expresses the gene at the right point in time and in
a cell- or tissue-specific manner. Usable promoters are
constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202),
such as those which originate from plant viruses, such as 35S CAMV
(Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also U.S.
Pat. No. 5,352,605 and WO 84/02913), 34S FMV (Sanger et al., Plant.
Mol. Biol., 14, 1990: 433-443), the parsley ubiquitin promoter, or
plant promoters such as the Rubisco small subunit promoter
described in U.S. Pat. No. 4,962,028 or the plant promoters PRP1
[Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, PGEL1, OCS
[Leisner (1988) Proc Natl Acad Sci USA 85(5): 2553-2557], lib4,
usp, mas [Comai (1990) Plant Mol Biol 15 (3):373-381], STLS1, ScBV
(Schenk (1999) Plant Mol Biol 39(6):1221-1230), B33, SAD1 or SAD2
(flax promoters, Jain et al., Crop Science, 39 (6), 1999:
1696-1701) or nos [Shaw et al. (1984) Nucleic Acids Res.
12(20):7831-7846]. Stable, constitutive expression of the proteins
according to the invention in a plant can be advantageous. However,
inducible expression of the polypeptide of the invention is
advantageous, if a late expression before the harvest is of
advantage, as metabolic manipulation may lead to plant growth
retardation.
[0378] The expression of plant genes can also be facilitated as
described above via a chemical inducible promoter (for a review,
see Gatz 1997, Annu Rev. Plant Physiol. Plant Mol. Biol.,
48:89-108). Chemically inducible promoters are particularly
suitable when it is desired to express the gene in a time-specific
manner. Examples of such promoters are a salicylic acid inducible
promoter (WO 95/19443), and abscisic acid-inducible promoter (EP
335 528), a tetracyclin-inducible promoter (Gatz et al. (1992)
Plant J. 2, 397-404), a cyclohexanol- or ethanol-inducible promoter
(WO 93/21334) or others as described herein.
[0379] Other suitable promoters are those which react to biotic or
abiotic stress conditions, for example the pathogen-induced PRP1
gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366),
the tomato heat-inducible hsp80 promoter (U.S. Pat. No. 5,187,267),
the potato chill-inducible alpha-amylase promoter (WO 96/12814) or
the wound-inducible pinII promoter (EP-A-0 375 091) or others as
described herein.
[0380] Preferred promoters are in particular those which bring
about gene expression in tissues and organs in which the
biosynthesis of metabolites takes place, in seed cells, such as
endosperm cells and cells of the developing embryo. Suitable
promoters are the oilseed rape napin gene promoter (U.S. Pat. No.
5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen
Genet, 1991, 225 (3): 459-67), the Arabidopsis oleosin promoter (WO
98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No.
5,504,200), the Brassica Bce4 promoter (WO 91/13980), the bean arcs
promoter, the carrot DcG3 promoter, or the Legumin B4 promoter
(LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9), and
promoters which bring about the seed-specific expression in
monocotyledonous plants such as maize, barley, wheat, rye, rice and
the like. Advantageous seed-specific promoters are the sucrose
binding protein promoter (WO 00/26388), the phaseolin promoter and
the napin promoter. Suitable promoters which must be considered are
the barley lpt2 or lpt1 gene promoter (WO 95/15389 and WO
95/23230), and the promoters described in WO 99/16890 (promoters
from the barley hordein gene, the rice glutelin gene, the rice
oryzin gene, the rice prolamin gene, the wheat gliadin gene, the
wheat glutelin gene, the maize zein gene, the oat glutelin gene,
the sorghum kasirin gene and the rye secalin gene). Further
suitable promoters are Amy32b, Amy 6-6 and Aleurain [U.S. Pat. No.
5,677,474], Bce4 (oilseed rape) [U.S. Pat. No. 5,530,149], glycinin
(soya) [EP 571 741], phosphoenolpyruvate carboxylase (soya) [JP
06/62870], ADR12-2 (soya) [WO 98/08962], isocitrate lyase (oilseed
rape) [U.S. Pat. No. 5,689,040] or .alpha.-amylase (barley) [EP 781
849]. Other promoters which are available for the expression of
genes in plants are leaf-specific promoters such as those described
in DE-A 19644478 or light-regulated promoters such as, for example,
the pea petE promoter.
[0381] Further suitable plant promoters are the cytosolic FBPase
promoter or the potato ST-LSI promoter (Stockhaus et al., EMBO J.
8, 1989, 2445), the Glycine max phosphoribosylpyrophosphate
amidotransferase promoter (GenBank Accession No. U87999) or the
node-specific promoter described in EP-A 0 249 676.
[0382] Other promoters, which are particularly suitable, are those
resulting in plastid-specific expression. Suitable promoters such
as the viral RNA polymerase promoter are described in WO 95/16783
and WO 97/06250, and the Arabidopsis clpP promoter, which is
described in WO 99/46394.
[0383] Other promoters, which are used for the strong expression of
nucleic acid sequences in as many tissues as possible, in
particular also in leaves, are, in addition to several of the
abovementioned viral and bacterial promoters, preferably, plant
promoters of actin or ubiquitin genes such as, for example, the
rice actin1 promoter. Further examples of constitutive plant
promoters are the sugarbeet V-ATPase promoters (WO 01/14572).
Examples of synthetic constitutive promoters are the Super promoter
(WO 95/14098) and promoters derived from G-boxes (WO 94/12015). If
appropriate, chemical inducible promoters may furthermore also be
used, compare EP-A 388 186, EP-A 335 528, WO 97/06268.
[0384] Another preferred embodiment of the invention is a nucleic
acid construct conferring the expression of the dsRNA molecule, the
antisense nucleic acid molecule, the ribozyme, the viral nucleic
acid molecule or the nucleic acid molecule as used in the inventive
process, suitable for the expression in plant.
[0385] Preferred recipient plants are, as described above, in
particular those plants, which can be transformed in a suitable
manner. These include monocotyledonous and dicotyledonous plants.
Plants which must be mentioned in particular are agriculturally
useful plants such as cereals and grasses, for example Triticum
spp., Zea mays, Hordeum vulgare, oats, Secale cereale, Oryza
sativa, Pennisetum glaucum, Sorghum bicolor, Triticale, Agrostis
spp., Cenchrus ciliaris, Dactylis glomerata, Festuca arundinacea,
Lolium spp., Medicago spp. and Saccharum spp., legumes and oil
crops, for example Brassica juncea, Brassica napus, Glycine max,
Arachis hypogaea, Gossypium hirsutum, Cicer arietinum, Helianthus
annuus, Lens culinaris, Linum usitatissimum, Sinapis alba,
Trifolium repens and Vicia narbonensis, vegetables and fruits, for
example bananas, grapes, Lycopersicon esculentum, asparagus,
cabbage, watermelons, kiwi fruit, Solanum tuberosum, Beta vulgaris,
cassava and chicory, trees, for example Coffea species, Citrus
spp., Eucalyptus spp., Picea spp., Pinus spp. and Populus spp.,
medicinal plants and trees, and flowers.
[0386] One embodiment of the present invention also relates to a
method for generating a vector, which comprises the insertion, into
a vector, of the nucleic acid molecule characterized herein, the
nucleic acid molecule according to the invention or the expression
cassette according to the invention. The vector can, for example,
be introduced into a cell, e.g. a microorganism or a plant cell, as
described herein for the nucleic acid construct, or below under
transformation or transfection or shown in the examples. A
transient or stable trans-formation of the host or target cell is
possible, however, a stable transformation is preferred.
[0387] The vector according to the invention is preferably a
vector, which is suitable for reducing, decreasing or deleting of
the polypeptide according to the invention in a plant. The method
can thus also encompass one or more steps for integrating
regulatory signals into the vector, in particular signals, which
mediate the reduction, decrease or deletion in a plant.
[0388] Accordingly, the present invention also relates to a vector
comprising the nucleic acid molecule characterized herein as part
of a nucleic acid construct suitable for plant expression or the
nucleic acid molecule according to the invention.
[0389] The advantageous vectors of the invention comprise the
nucleic acid molecules which encode proteins according to the
invention, nucleic acid molecules which are used in the process, or
nucleic acid construct suitable for the expression in plants as
described above comprising the nucleic acid molecules used, either
alone or in combination with further genes such as the biosynthesis
or regulatory genes of plant metabolisms e.g. with the genes
mentioned herein above. In accordance with the invention, the term
"vector" refers to a nucleic acid molecule, which is capable of
transporting another nucleic acid to which it is linked. One type
of vector is a "plasmid", which means a circular double-stranded
DNA loop into which additional DNA segments can be ligated. A
further type of vector is a viral vector, it being possible to
ligate additional DNA segments into the viral genome. Certain
vectors are capable of autonomous replication in a host cell into
which they have been introduced (for example bacterial vectors with
bacterial replication origin). Other preferred vectors are
advantageously completely or partly integrated into the genome of a
host cell when they are introduced into the host cell and thus
replicate together with the host genome. Moreover, certain vectors
are capable of controlling the expression of genes with which they
are in operable linkage. In the present context, these vectors are
referred to as "expression vectors". As mentioned above, they are
capable of autonomous replication or may be integrated partly or
completely into the host genome. Expression vectors, which are
suitable for DNA recombination techniques usually, take the form of
plasmids. In the present description, "plasmid" and "vector" can be
used interchangeably since the plasmid is the most frequently used
form of a vector. However, the invention is also intended to
encompass these other forms of expression vectors, such as viral
vectors, which exert similar functions. The term vector is
furthermore also to encompass other vectors which are known to the
skilled worker, such as phages, viruses such as SV40, CMV, TMV,
transposons, IS elements, phasmids, phagemids, cosmids, and linear
or circular DNA.
[0390] The recombinant expression vectors which are advantageously
used in the process comprise the nucleic acid molecules according
to the invention or the nucleic acid construct according to the
invention in a form which is suitable for repressing the nucleic
acid molecules of the invention and/or in the same time expressing,
in a host cell, additional genes, which are accompanied by the
nucleic acid molecules according to the invention or described
herein. Accordingly, the recombinant expression vectors comprise
one or more regulatory signals selected on the basis of the host
cells to be used for the expression, in operable linkage with the
nucleic acid sequence to be expressed.
[0391] In a recombinant expression vector, "operable linkage" means
that the nucleic acid molecule of interest is linked to the
regulatory signals in such a way that expression of the genes is
possible: they are linked to one another in such a way that the two
sequences fulfill the predicted function assigned to the sequence
(for example in an in-vitro transcription/translation system, or in
a host cell if the vector is introduced into the host cell).
[0392] The term "regulatory sequence" is intended to comprise
promoters, enhancers and other expression control elements (for
example polyadenylation signals). These regulatory sequences are
described, for example, in Goeddel: Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990), or see: Gruber and Crosby, in: Methods in Plant Molecular
Biology and Biotechnology, CRC Press, Boca Raton, Fla., Ed.: Glick
and Thompson, chapter 7, 89-108, including the references cited
therein. Regulatory sequences encompass those, which control the
constitutive expression of a nucleotide sequence in many types of
host cells and those which control the direct expression of the
nucleotide sequence in specific host cells only, and under specific
conditions. The skilled worker knows that the design of the
expression vector may depend on factors such as the selection of
the host cell to be transformed, the extent to which the desired
protein is expressed, and the like. A preferred selection of
regulatory sequences is described above, for example promoters,
terminators, enhancers and the like. The term regulatory sequence
is to be considered as being encompassed by the term regulatory
signal. Several advantageous regulatory sequences, in particular
promoters and terminators are described above. In general, the
regulatory sequences described as advantageous for nucleic acid
construct suitable for expression are also applicable for
vectors.
[0393] The recombinant expression vectors used can be designed
specifically for the expression, in prokaryotic and/or eukaryotic
cells, of nucleic acid molecules used in the process. This is
advantageous since intermediate steps of the vector construction
are frequently carried out in microorganisms for the sake of
simplicity. For example, the genes according to the invention and
other genes can be expressed in bacterial cells, insect cells
(using baculovirus expression vectors), yeast cells and other
fungal cells [Romanos (1992), Yeast 8:423-488; van den Hondel,
(1991), in: More Gene Manipulations in Fungi, J. W. Bennet & L.
L. Lasure, Ed., pp. 396-428: Academic Press: San Diego; and van den
Hondel, C. A. M. J. J. (1991), in: Applied Molecular Genetics of
Fungi, Peberdy, J. F., et al., Ed., pp. 1-28, Cambridge University
Press: Cambridge], algae [Falciatore et al., 1999, Marine
Biotechnology. 1, 3:239-251] using vectors and following a
transformation method as described in WO 98/01572, and preferably
in cells of multi-celled plants [see Schmidt, R. and Willmitzer, L.
(1988) Plant Cell Rep.:583-586; Plant Molecular Biology and
Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, pp. 71-119
(1993); F. F. White, in: Transgenic Plants, Bd. 1, Engineering and
Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-43;
Potrykus, Annu Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),
205-225 (and references cited therein)]. Suitable host cells are
furthermore discussed in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). As an alternative, the sequence of the recombinant
expression vector can be transcribed and translated in vitro, for
example using T7 promotor-regulatory sequences and T7
polymerase.
[0394] In most cases, proteins can be expressed in prokaryotes
using vectors comprising constitutive or inducible promoters, which
control the expression of fusion proteins or nonfusion proteins.
Typical fusion expression vectors are, inter alia, pGEX (Pharmacia
Biotech Inc; Smith, D. B., and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRITS
(Pharmacia, Piscataway, N.J.), in which glutathione-S-transferase
(GST), maltose-E-binding protein or protein A is fused with the
recombinant target protein. Examples of suitable inducible
nonfusion E. coli expression vectors are, inter alia, pTrc (Amann
et al. (1988) Gene 69:301-315) and pET 11d [Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 60-89]. The target gene expression of the
pTrc vector is based on the transcription of a hybrid trp-lac
fusion promoter by the host RNA polymerase. The target gene
expression from the pET 11d vector is based on the transcription of
a T7-gn10-lac fusion promoter, which is mediated by a coexpressed
viral RNA polymerase (T7 gn1). This viral polymerase is provided by
the host strains BL21 (DE3) or HMS174 (DE3) by a resident
1-prophage, which harbors a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0395] Other vectors which are suitable in prokaryotic organisms
are known to the skilled worker; these vectors are for example in
E. coli pLG338, pACYC184, the pBR series, such as pBR322, the pUC
series such as pUC18 or pUC19, the M113 mp series, pKC30, pRep4,
pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III.sup.113-B1,
lgt11 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361,
in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or
pAJ667.
[0396] In a further embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in the yeasts
S. cerevisiae encompass pYeDesaturasec1 (Baldari et al. (1987) Embo
J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943),
pJRY88 (Schultz et al. (1987) Gene 54:113-123) and pYES2
(Invitrogen Corporation, San Diego, Calif.). Vectors and methods
for the construction of vectors which are suitable for use in other
fungi, such as the filamentous fungi, encompass those which are
described in detail in: van den Hondel, C. A. M. J. J. [(1991), J.
F. Peberdy, Ed., pp. 1-28, Cambridge University Press: Cambridge;
or in: More Gene Manipulations in Fungi; J. W. Bennet & L. L.
Lasure, Ed., pp. 396-428: Academic Press: San Diego]. Examples of
other suitable yeast vectors are 2 .mu.M, pAG-1, YEp6, YEp13 or
pEMBLYe23.
[0397] Further vectors, which may be mentioned by way of example,
are pALS1, pIL2 or pBB116 in fungi or pLGV23, pGHlac.sup.+, pBIN19,
pAK2004 or pDH51 in plants.
[0398] The abovementioned vectors are only a small overview of
potentially suitable vectors. Further plasmids are known to the
skilled worker and are described, for example, in: Cloning Vectors
(Ed. Pouwels, P. H., et al., Elsevier, Amsterdam-New York-Oxford,
1985, ISBN 0 444 904018). Further suitable expression systems for
prokaryotic and eukaryotic cells, see the chapters 16 and 17 by
Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning:
A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989.
[0399] Accordingly, one embodiment of the invention relates to a
vector comprising the nucleic acid molecule according to the
invention or a nucleic acid construct of the invention. Said vector
is useful for the reduction, decrease or deletion of the
polypeptide according to the invention in an organism preferably a
plant. Advantageously the nucleic acid molecule of the invention is
in an operable linkage with regulatory sequences for the expression
in a prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic
host. Furthermore vectors suitable for homologous recombination are
also within the scope of the invention.
[0400] Accordingly, one embodiment of the invention relates to a
host cell, which has been transformed stably or transiently with
the vector according to the invention or the nucleic acid molecule
according to the invention or the nucleic acid construct according
to the invention. Said host cell is preferably a plant cell.
[0401] The skilled worker knows that protein and DNA expressed in
different organisms differ in many respects and properties, e.g.
methylation, degradation and post-translational modification as for
example glucosylation, phosphorylation, acetylation,
myristoylation, ADP-ribosylation, farnesylation, carboxylation,
sulfation, ubiquination, etc. though having the same coding
sequence. Preferably, the cellular expression control of the
corresponding protein differs accordingly in the control mechanisms
controlling the activity and expression of an endogenous protein or
another eukaryotic protein.
[0402] In one embodiment, the present invention relates to a
polypeptide having the biological activity represented by the
protein of the invention. In one embodiment, said polypeptide
having the biological activity represented by the protein of the
invention distinguishes over the sequence depicted in SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ
ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:
48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ
ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108,
SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ
ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID
NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288 by one or more amino acids. In another
embodiment, said polypeptide of the invention does not consist of
the sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ
ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:
16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ
ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:
34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ
ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO:
52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ
ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO:
70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ
ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO:
104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288. In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:
44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ
ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:
62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ
ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:
80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104,
SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ
ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID
NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288.
[0403] In one embodiment, the present invention relates to a
polypeptide having the amino acid sequence encoded by a nucleic
acid molecule of the invention or obtainable by a process of the
invention. Said polypeptide confers preferably the aforementioned
activity, in particular, the polypeptide confers the increase of
plant growth in a cell, tissue or a plant or a part thereof after
decreasing the cellular activity, e.g. by decreasing the expression
or the specific activity of the polypeptide. In one embodiment,
said polypeptide distinguishes over the sequence depicted in SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:
20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ
ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:
38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ
ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:
56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ
ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:
74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ
ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID
NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288 by one or more amino acids.
Preferably, the sequence of the polypeptide of the invention
distinguishes from the sequence shown in SEQ ID NO: 2, SEQ ID NO:
4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID
NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID
NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID
NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:
110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO:
118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:
126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288 by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In one
embodiment, polypeptide distinguishes form the sequence shown in
SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID
NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,
SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID
NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,
SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID
NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,
SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID
NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO:
106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 by more than
5, 6, 7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25
or 30 amino acids, even more preferred are more than 40, 50, or 60
amino acids. In another embodiment, said polypeptide of the
invention does not consist of the sequence shown in SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ
ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:
30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ
ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:
48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ
ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:
66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ
ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:
84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108,
SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ
ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID
NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:
134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:
142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:
150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:
158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO:
190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO:
198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO:
206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO:
214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO:
222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO:
230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO:
238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO:
246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO:
270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO:
278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286
or SEQ ID NO: 288. In a further embodiment, said polypeptide of the
present invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical.
[0404] The terms "protein" and "polypeptide" used in this
application are interchangeable. "Polypeptide" refers to a polymer
of amino acids (amino acid sequence) and does not refer to a
specific length of the molecule. Thus peptides and oligopeptides
are included within the definition of polypeptide. This term does
also refer to or include posttranslational modifications of the
polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like. Included within the definition are,
for example, polypeptides containing one or more analogs of an
amino acid (including, for example, unnatural amino acids, etc.),
polypeptides with substituted linkages, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring.
[0405] Preferably, the polypeptide is isolated. An "isolated" or
"purified" protein or nucleic acid molecule or biologically active
portion thereof is substantially free of cellular material when
produced by recombinant DNA techniques, or chemical precursors or
other chemicals when chemically synthesized.
[0406] A polypeptide of the invention can participate in the
process of the present invention. The polypeptide or a portion
thereof comprises preferably an amino acid sequence which is
sufficiently homologous to an amino acid sequence shown in SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:
20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ
ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:
38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ
ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:
56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ
ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:
74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ
ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID
NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288 such that the protein or
portion thereof maintains the ability to confer the activity of the
present invention, that means an increase in plant growth by
decreasing its biological activity. Preferably, the polypeptide
used in the inventive process has an amino acid sequence identical
as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288.
[0407] Further, the polypeptide can have an amino acid sequence
which is encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions as described
above, to a nucleotide sequence of the nucleic acid molecule of the
present invention. Accordingly, the polypeptide has an amino acid
sequence which is encoded by a nucleotide sequence that is at least
about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least
about 75%, 80%, 85% or 90%, and more preferably at least about 91%,
92%, 93%, 94% or 95%, and even more preferably at least about 96%,
97%, 98%, 99% or more homologous to one of the nucleic acid
sequences acids SEQ ID 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID
NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,
SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,
SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID
NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID
NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,
SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID
NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO:
113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:
185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO:
193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO:
201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO:
225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO:
233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO:
241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO:
249, SEQ ID NO: 251, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO:
273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:
281, SEQ ID NO: 283, SEQ ID NO: 285 or SEQ ID NO: 287. The
preferred polypeptide of the present invention preferably possesses
at least one of the activities according to the invention and
described herein. A preferred polypeptide of the present invention
includes an amino acid sequence encoded by a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions,
to a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 113 or which
is homologous thereto, as defined above.
[0408] Accordingly the polypeptide of the present invention can
vary from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:
114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:
122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288 in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID
NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,
SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID
NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,
SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID
NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID
NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,
SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID
NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86,
SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ
ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID
NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:
168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:
176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:
184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:
192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO:
200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:
208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO:
216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:
224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO:
232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO:
240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO:
248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO:
272, SEQ ID NO: 274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO:
280, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO:
288.
[0409] For the comparison of amino acid sequences the same
algorithms as described above or nucleic acid sequences can be
used. Results of high quality are reached by using the algorithm of
Needleman and Wunsch or Smith and Waterman. Therefore programs
based on said algorithms are preferred. Advantageously the
comparisons of sequences can be done with the program PileUp (J.
Mol. Evolution, 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989:
151-153) or preferably with the programs Gap and BestFit, which are
respectively based on the algorithms of Needleman and Wunsch [J.
Mol. Biol. 48; 443-453 (1970)] and Smith and Waterman [Adv. Appl.
Math. 2; 482-489 (1981)]. Both programs are part of the GCG
software-package [Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711 (1991); Altschul et al. (1997) Nucleic
Acids Res. 25:3389 et seq.]. Therefore preferably the calculations
to determine the perentages of sequence homology are done with the
program Gap over the whole range of the sequences. The following
standard adjustments for the comparison of amino acid sequences
were used: gap weight: 8, length weight: 2, average match: 2.912,
average mismatch: -2.003.
[0410] Biologically active portions of an polypeptide of the
present invention include peptides comprising amino acid sequences
derived from the amino acid sequence of the polypeptide of the
present invention, e.g., the amino acid sequence shown in SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:
20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ
ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:
38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ
ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:
56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ
ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:
74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ
ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID
NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:
164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO:
172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO:
180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO:
188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:
196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO:
204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO:
212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO:
220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO:
228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO:
236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO:
244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO:
252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO: 274, SEQ ID NO:
276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO: 282, SEQ ID NO:
284, SEQ ID NO: 286 or SEQ ID NO: 288 or the amino acid sequence of
a protein homologous thereto, which include fewer amino acids than
a full length protein having the biological activity of the protein
of the invention or the polypeptide of the present invention or the
full length protein which is homologous to a protein having the
biological activity of the protein of the invention or the
polypeptide of the present invention depicted herein, and exhibit
at least one activity of the polypeptide of the present invention,
which leads to an increase in yield after reduction of its
expression or activity. Typically, biologically (or
immunologically) active portions i.e. peptides, e.g., peptides
which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40,
50, 100 or more amino acids in length comprise a domain or motif
with at least one activity or epitope of a or of the polypeptide of
the present invention. Moreover, other biologically active
portions, in which other regions of the polypeptide are deleted,
can be prepared by recombinant techniques and evaluated for one or
more of the activities described herein.
[0411] Any mutagenesis strategies for the polypeptide of the
present invention, which result in a decreasing biological activity
disclosed herein are not meant to be limiting; variations on these
strategies will be readily apparent to one skilled in the art.
Using such strategies, and incorporating the mechanisms disclosed
herein, the nucleic acid molecule and polypeptide of the invention
may be utilized to generate plants or parts thereof, expressing
mutated nucleic acid molecule and/or polypeptide molecules of the
invention such that the yield, production, and/or efficiency of
production of a desired compound such as the fine chemical is
improved. This desired compound may be any natural product of
plants, which includes the final products of biosynthesis pathways
and intermediates of naturally-occurring metabolic pathways, as
well as molecules which do not naturally occur in the metabolism of
said cells, but which are produced by a said cells of the
invention.
[0412] Furthermore, folding simulations and computer redesign of
structural motifs of the protein of the invention can be performed
using appropriate computer programs (Olszewski, Proteins 25 (1996),
286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679).
Computer modeling of protein folding can be used for the
conformational and energetic analysis of detailed peptide and
protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf,
Adv. Exp. Med. Biol. 376 (1995), 37-45). The appropriate programs
can be used for the identification of interactive sites of the
polypeptide of the invention and its substrates or binding factors
or other interacting proteins by computer assistant searches for
complementary peptide sequences (Fassina, Immunomethods (1994),
114-120). Further appropriate computer systems for the design of
protein and peptides are described in the prior art, for example in
Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y.
Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986),
5987-5991. The results obtained from the above-described computer
analysis can be used for, e.g., the preparation of peptidomimetics
of the protein of the invention or fragments thereof. Such
pseudopeptide analogues of the, natural amino acid sequence of the
protein may very efficiently mimic the parent protein (Benkirane,
J. Biol. Chem. 271 (1996), 33218-33224). For example, incorporation
of easily available achiral Q-amino acid residues into a protein of
the invention or a fragment thereof results in the substitution of
amide bonds by polymethylene units of an aliphatic chain, thereby
providing a convenient strategy for constructing a peptidomimetic
(Banerjee, Biopolymers 39 (1996), 769-777).
[0413] Superactive peptidomimetic analogues of small peptide
hormones in other systems are described in the prior art (Zhang,
Biochem. Biophys. Res. Commun. 224 (1996), 327-331). Appropriate
peptidomimetics of the protein of the present invention can also be
identified by the synthesis of peptidomimetic combinatorial
libraries through successive amide alkylation and testing the
resulting compounds, e.g., for their binding and immunological
properties. Methods for the generation and use of peptidomimetic
combinatorial libraries are described in the prior art, for example
in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner,
Bioorg. Med. Chem. 4 (1996), 709-715.
[0414] Furthermore, a three-dimensional and/or crystallographic
structure of the protein of the invention can be used for the
design of peptidomimetic inhibitors of the biological activity of
the protein of the invention (Rose, Biochemistry 35 (1996),
12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).
[0415] Furthermore, a three-dimensional and/or crystallographic
structure of the protein of the invention and the identification of
interactive sites the polypeptide of the invention and its
substrates or binding factors can be used for design of mutants
with modulated binding or turn over activities. For example, the
active center of the polypeptide of the present invention can be
modelled and amino acid residues participating in the catalytic
reaction can be modulated to increase or decrease the binding of
the substrate to inactivate the polypeptide. The identification of
the active center and the amino acids involved in the catalytic
reaction facilitates the screening for mutants having an increased
activity.
[0416] The sequence shown in SEQ ID NO: 2 has been described under
its Accession Number At5g64410 or NP.sub.--201246 as a protein of
the OPT oligopeptide transporter protein family. Homologues of this
protein are depicted in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 104, SEQ ID
NO: 106, SEQ ID NO: 108, SEQ ID NO: 110 or SEQ ID NO: 112. The
proteins contain a protein domain which is identical or similar to
the SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or
SEQ ID NO: 91 domain.
[0417] The sequence shown in SEQ ID NO: 114 has been described
under its Accession Number At5g02270 as a protein of the ABC
transporter family. Homologues of this protein are depicted in SEQ
ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID
NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO:
130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:
138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:
146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:
154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO:
162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:
186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO:
194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO:
202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:
210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO:
218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO:
226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO:
234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO:
242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO:
250, SEQ ID NO: 252, SEQ ID NO: 270, SEQ ID NO: 272, SEQ ID NO:
274, SEQ ID NO: 276, SEQ ID NO: 278, SEQ ID NO: 280, SEQ ID NO:
282, SEQ ID NO: 284, SEQ ID NO: 286 or SEQ ID NO: 288. The proteins
contain a protein domain which is identical or similar to the SEQ
ID NO: 265, SEQ ID NO: 266, SEQ ID NO:267 or SEQ ID NO: 268
domain.
[0418] One embodiment of the invention also relates to an antibody,
which binds specifically to the polypeptide according to the
invention or parts, i.e. specific fragments or epitopes of such a
protein.
[0419] The antibodies of the invention can be used to identify and
isolate the polypeptide according to the invention and encoding
genes in any organism, preferably plants, prepared in plants
described herein. Such antibodies can also be expressed in the
suitable host organisms thereby reducing the biological activity of
the genes of the invention by binding to their protein products
leading for example to a steric interferance with their biological
activity. These antibodies can be monoclonal antibodies, polyclonal
antibodies or synthetic anti-bodies as well as fragments of
antibodies, such as Fab, Fv or scFv fragments etc. Monoclonal
antibodies can be prepared, for example, by the techniques as
originally described in Kohler and Milstein, Nature 256 (1975),
495, and Galfr6, Meth. Enzymol. 73 (1981), 3, which comprise the
fusion of mouse myeloma cells to spleen cells derived from
immunized mammals.
[0420] Furthermore, antibodies or fragments thereof to the
aforementioned peptides can be obtained by using methods, which are
described, e.g., in Harlow and Lane "Antibodies, A Laboratory
Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can
be used, for example, for the immunoprecipitation and
immunolocalization of proteins according to the invention as well
as for the monitoring of the synthesis of such proteins, for
example, in recombinant organisms, and for the identification of
compounds interacting with the protein according to the invention.
For example, surface plasmon resonance as employed in the BIAcore
system can be used to increase the efficiency of phage antibodies
selections, yielding a high increment of affinity from a single
library of phage antibodies, which bind to an epitope of the
protein of the invention (Schier, Human Antibodies Hybridomas 7
(1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In
many cases, the binding phenomena of antibodies to antigens is
equivalent to other ligand/anti-ligand binding.
[0421] A further embodiment of the invention also relates to a
method for the generation of a transgenic host cell, e.g. a
eukaryotic or prokaryotic host or host cell, preferably a
transgenic microorganism, a transgenic plant cell or a transgenic
plant tissue or a transgenic plant, which comprises introducing,
into the plant, the plant cell or the plant tissue, the nucleic
acid construct according to the invention, the vector according to
the invention, or the nucleic acid molecule according to the
invention.
[0422] A further embodiment of the invention also relates to a
method for the transient generation of a host or host cell,
eukaryotic or prokaryotic cell, preferably a transgenic plant cell
or a transgenic plant tissue or a transgenic plant, which comprises
introducing, into the plant, the plant cell or the plant tissue,
the nucleic acid construct according to the invention, the vector
according to the invention, the nucleic acid molecule characterized
herein as being contained in the nucleic acid construct of the
invention or the nucleic acid molecule according to the invention,
whereby the introduced nucleic acid molecules, nucleic acid
construct and/or vector is not integrated into the genome of the
host or host cell. Therefore the transformants are not stable
during the propagation of the host in respect of the introduced
nucleic acid molecules, nucleic acid construct and/or vector.
[0423] In the process according to the invention, transgenic
organisms are also to be understood as meaning--if they take the
form of plants--plant cells, plant tissues, plant organs such as
root, shoot, stem, seed, flower, tuber or leaf, or intact
plants.
[0424] Growing is to be understood as meaning for example culturing
the transgenic plant cells, plant tissue or plant organs on or in a
nutrient medium or the intact plant on or in a substrate, for
example in hydroponic culture, potting compost or on a field
soil.
[0425] In a further advantageous embodiment of the process, the
nucleic acid molecules can be expressed in single-celled plant
cells (such as algae), see Falciatore et al., 1999, Marine
Biotechnology 1 (3): 239-251 and references cited therein, and
plant cells from higher plants (for example spermatophytes such as
crops). Examples of plant expression vectors encompass those which
are described in detail herein or in: Becker, D. [(1992) Plant Mol.
Biol. 20:1195-1197] and Bevan, M. W. [(1984), Nucl. Acids Res.
12:8711-8721; Vectors for Gene Transfer in Higher Plants; in:
Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung
and R. Wu, Academic Press, 1993, pp. 15-38]. An overview of binary
vectors and their use is also found in Hellens, R. [(2000), Trends
in Plant Science, Vol. 5 No. 10, 446-451.
[0426] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
The terms "transformation" and "transfection" include conjugation
and transduction and, as used in the present context, are intended
to encompass a multiplicity of prior-art methods for introducing
foreign nucleic acid molecules (for example DNA) into a host cell,
including calcium phosphate coprecipitation or calcium chloride
coprecipitation, DEAE-dextran-mediated transfection, PEG-mediated
transfection, lipofection, natural competence, chemically mediated
transfer, electroporation or particle bombardment. Suitable methods
for the transformation or transfection of host cells, including
plant cells, can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual., 2'' Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and
in other laboratory handbooks such as Methods in Molecular Biology,
1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey,
Humana Press, Totowa, N.J.
[0427] The above-described methods for the transformation and
regeneration of plants from plant tissues or plant cells are
exploited for transient or stable transformation of plants.
Suitable methods are the transformation of protoplasts by
polyethylene-glycol-induced DNA uptake, the biolistic method with
the gene gun--known as the particle bombardment method-,
electroporation, the incubation of dry embryos in DNA-containing
solution, microinjection and the Agrobacterium-mediated gene
transfer. The abovementioned methods are described for example in
B. Jenes, Techniques for Gene Transfer, in: Transgenic Plants, Vol.
1, Engineering and Utilization, edited by S. D. Kung and R. Wu,
Academic Press (1993) 128-143 and in Potrykus Annu Rev. Plant
Physiol. Plant Molec. Biol. 42 (1991) 205-225. The construct to be
expressed is preferably cloned into a vector, which is suitable for
transforming Agrobacterium tumefaciens, for example pBin19 (Bevan,
Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed with
such a vector can then be used in the known manner for the
transformation of plants, in particular crop plants, such as, for
example, tobacco plants, for example by bathing scarified leaves or
leaf segments in an agrobacterial solution and subsequently
culturing them in suitable media. The transformation of plants with
Agrobacterium tumefaciens is described for example by Hofgen and
Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or known from, inter
alia, F. F. White, Vectors for Gene Transfer in Higher Plants; in
Transgenic Plants, Vol. 1, Engineering and Utilization, edited by
S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
[0428] To select for the successful transfer of the nucleic acid
molecule, vector or nucleic acid construct of the invention
according to the invention into a host organism, it is advantageous
to use marker genes as have already been described above in detail.
It is known of the stable or transient integration of nucleic acids
into plant cells that only a minority of the cells takes up the
foreign DNA and, if desired, integrates it into its genome,
depending on the expression vector used and the transfection
technique used. To identify and select these integrants, a gene
encoding for a selectable marker (as described above, for example
resistance to antibiotics) is usually introduced into the host
cells together with the gene of interest. Preferred selectable
markers in plants comprise those, which confer resistance to an
herbicide such as glyphosate or gluphosinate. Other suitable
markers are, for example, markers, which encode genes involved in
biosynthetic pathways of, for example, sugars or amino acids, such
as .beta.-galactosidase, ura3 or ilv2. Markers, which encode genes
such as luciferase, gfp or other fluorescence genes, are likewise
suitable. These markers and the aforementioned markers can be used
in mutants in whom these genes are not functional since, for
example, they have been deleted by conventional methods.
Furthermore, nucleic acid molecules, which encode a selectable
marker, can be introduced into a host cell on the same vector as
those, which encode the polypeptides of the invention or used in
the process or else in a separate vector. Cells which have been
transfected stably with the nucleic acid introduced can be
identified for example by selection (for example, cells which have
integrated the selectable marker survive whereas the other cells
die).
[0429] Since the marker genes, as a rule specifically the gene for
resistance to antibiotics and herbicides, are no longer required or
are undesired in the transgenic host cell once the nucleic acids
have been introduced successfully, the process according to the
invention for introducing the nucleic acids advantageously employs
techniques which enable the removal, or excision, of these marker
genes. One such a method is what is known as cotransformation. The
cotransformation method employs two vectors simultaneously for the
transformation, one vector bearing the nucleic acid according to
the invention and a second bearing the marker gene(s). A large
proportion of transformants receives or, in the case of plants,
comprises (up to 40% of the transformants and above), both vectors.
The marker genes can subsequently be removed from the transformed
plant by performing crosses. In another method, marker genes
integrated into a transposon are used for the transformation
together with desired nucleic acid (known as the Ac/Ds technology).
In some cases (approx. 10%), the transposon jumps out of the genome
of the host cell once transformation has taken place successfully
and is lost. In a further number of cases, the transposon jumps to
a different location. In these cases, the marker gene must be
eliminated by performing crosses. In microbiology, techniques were
developed which make possible, or facilitate, the detection of such
events. A further advantageous method relies on what are known as
recombination systems, whose advantage is that elimination by
crossing can be dispensed with. The best-known system of this type
is what is known as the Cre/lox system. Cre1 is a recombinase,
which removes the sequences located between the loxP sequence. If
the marker gene is integrated between the loxP sequence, it is
removed, once transformation has taken place successfully, by
expression of the recombinase. Further recombination systems are
the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol.
Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol.,
149, 2000: 553-566). A site-specific integration into the plant
genome of the nucleic acid sequences according to the invention is
possible. Naturally, these methods can also be applied to
microorganisms such as yeast, fungi or bacteria.
[0430] Agrobacteria transformed with an expression vector according
to the invention may also be used in the manner known per se for
the transformation of plants such as experimental plants like
Arabidopsis or crop plants, such as, for example, cereals, maize,
oats, rye, barley, wheat, soya, rice, cotton, sugarbeet, canola,
sunflower, flax, hemp, potato, tobacco, tomato, carrot, bell
peppers, oilseed rape, tapioca, cassava, arrow root, tagetes,
alfalfa, lettuce and the various tree, nut, and grapevine species,
in particular oil-containing crop plants such as soya, peanut,
castor-oil plant, sunflower, maize, cotton, flax, oilseed rape,
coconut, oil palm, safflower (Carthamus tinctorius) or cocoa beans,
for example by bathing scarified leaves or leaf segments in an
agrobacterial solution and subsequently growing them in suitable
media.
[0431] In addition to the transformation of somatic cells, which
then has to be regenerated into intact plants, it is also possible
to transform the cells of plant meristems and in particular those
cells which develop into gametes. In this case, the transformed
gametes follow the natural plant development, giving rise to
transgenic plants. Thus, for example, seeds of Arabidopsis are
treated with agrobacteria and seeds are obtained from the
developing plants of which a certain proportion is transformed and
thus transgenic (Feldman, K A and Marks M D (1987). Mol Gen Genet.
208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell,
eds, Methods in Arabidopsis Research. Word Scientific, Singapore,
pp. 274-289). Alternative methods are based on the repeated removal
of the influorescences and incubation of the excision site in the
center of the rosette with transformed agrobacteria, whereby
transformed seeds can likewise be obtained at a later point in time
(Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet,
245: 363-370). However, an especially effective method is the
vacuum infiltration method with its modifications such as the
"floral dip" method. In the case of vacuum infiltration of
Arabidopsis, intact plants under reduced pressure are treated with
an agrobacterial suspension (Bechthold, N (1993). C R Acad Sci
Paris Life Sci, 316: 1194-1199), while in the case of the"floral
dip" method the developing floral tissue is incubated briefly with
a surfactant-treated agrobacterial suspension (Clough, S J and
Bent, A F (1998). The Plant J. 16, 735-743). A certain proportion
of transgenic seeds are harvested in both cases, and these seeds
can be distinguished from nontransgenic seeds by growing under the
above-described selective conditions.
[0432] The genetically modified plant cells can be regenerated via
all methods with which the skilled worker is familiar. Suitable
methods can be found in the above-mentioned publications by S. D.
Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
[0433] Accordingly, the present invention thus also relates to a
plant cell comprising the nucleic acid construct according to the
invention, the nucleic acid molecule according to the invention or
the vector according to the invention.
[0434] Accordingly the present invention relates to any cell
transgenic for any nucleic acid characterized as part of the
invention, e.g. conferring the increase in plant growth in a cell
or an organism or a part thereof, e.g. the nucleic acid molecule of
the invention, the nucleic acid construct of the invention, the
antisense molecule of the invention, the vector of the invention or
a nucleic acid molecule encoding the polypeptide of the invention,
e.g. encoding a polypeptide having biological activity of the
protein of the invention. Due to the abovementioned activity the
growth in a plant cell or a plant is increased. For example, due to
modulation or manupulation, the cellular activity is increased,
e.g. due to a decreased expression or decreased specific activity
of the subject matters of the invention in a plant cell or a plant
or a plant part thereof.
[0435] A naturally occurring expression cassette--for example the
naturally occurring combination of the promoter of the protein of
the invention with the corresponding gene, which codes for the
protein of the invention--becomes a transgenic expression cassette
when it is modified by non-natural, synthetic "artificial" methods
such as, for example, mutagenization. Such methods have been
described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[0436] Further, the plant cell, plant tissue or plant can also be
transformed such that further enzymes and proteins are
(over)expressed which expression supports an increase in plant
growth. Similarly the plant cell, plant tissue or plant can also be
transformed such that further enzymes, transporters or other
proteins are inhibited in its expression or activity leading to a
further increase in plant growth
[0437] The term "transgenic plants" used in accordance with the
invention refers to the progeny of a transgenic plant, for example
the T.sub.1, T.sub.2, T.sub.3 and subsequent plant generations or
the BC.sub.1, BC.sub.2, BC.sub.3 and subsequent plant generations.
Thus, the transgenic plants according to the invention can be
raised and selfed or crossed with other individuals in order to
obtain further transgenic plants according to the invention.
Transgenic plants may also be obtained by propagating transgenic
plant cells vegetatively. The present invention also relates to
trans-genic plant material, which can be derived from a transgenic
plant population according to the invention. Such material includes
plant cells and certain tissues, organs and parts of plants in all
their manifestations, such as seeds, leaves, anthers, fibers,
tubers, roots, root hairs, stems, embryo, calli, cotelydons,
petioles, harvested material, plant tissue, reproductive tissue and
cell cultures, which are derived from the actual transgenic plant
and/or can be used for bringing about the transgenic plant.
[0438] Any transformed plant obtained according to the invention
can be used in a conventional breeding scheme or in in vitro plant
propagation to produce more transformed plants with the same
characteristics and/or can be used to introduce the same
characteristic in other varieties of the same or related species.
Such plants are also part of the invention. Seeds obtained from the
transformed plants genetically also contain the same characteristic
and are part of the invention. As mentioned before, the present
invention is in principle applicable to any plant and crop that can
be transformed with any of the transformation method known to those
skilled in the art.
[0439] In an especially preferred embodiment, the host cell, plant
cell, plant or plant tissue according to the invention is
transgenic.
[0440] Accordingly, the invention therefore relates to transgenic
plants transformed with at least one nucleic acid molecule, nucleic
acid construct or vector according to the invention, and to plant
cells, plant cell cultures, plant tissues, plant parts--such as,
for example leaves, roots and the like--or propagation material
derived from such plants. The terms "recombinant (host)" and
"transgenic (host)" are used interchangeably in this context.
Naturally, these terms refer not only to the host organism or
target cell in question, but also to the progeny, or potential
progeny, of these plants or plant cells. Since certain
modifications may occur in subsequent generations owing to mutation
or environmental effects, such progeny is not necessarily identical
with the parental cell, but still comes within the scope of the
term as used herein.
[0441] Suitable plants for the process according to the invention
or as hosts are e.g. crop plants. The plants used as hosts are
plants, such as dictotyledonous or monocotyledonous plants.
[0442] In principle all plants can be used as host organism,
especially the plants mentioned above as source organism. Preferred
transgenic plants are, for example, selected from the families
Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae,
Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Malvaceae,
Nymphaeaceae, Papaveraceae, Rosaceae, Salicaceae, Solanaceae,
Arecaceae, Bromeliaceae, Cyperaceae, Iridaceae, Liliaceae,
Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae, Ranunculaceae,
Carifolaceae, Rubiaceae, Scrophulariaceae, Caryophyllaceae,
Ericaceae, Polygonaceae, Violaceae, Juncaceae or Poaceae and
preferably from a plant selected from the group of the families
Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae,
Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Preferred
are crop plants such as plants advantageously selected from the
group of the genus peanut, oilseed rape, canola, sunflower,
safflower, olive, sesame, hazelnut, almond, avocado, bay,
pumpkin/squash, linseed, soya, pistachio, borage, maize, wheat,
rye, oats, sorghum and millet, triticale, rice, barley, cassava,
potato, sugarbeet, egg plant, alfalfa, and perennial grasses and
forage plants, oil palm, vegetables (brassicas, root vegetables,
tuber vegetables, pod vegetables, fruiting vegetables, onion
vegetables, leafy vegetables and stem vegetables), buckwheat,
Jerusalem artichoke, broad bean, vetches, lentil, dwarf bean,
lupin, clover and Lucerne for mentioning only some of them.
[0443] Preferred plant cells, plant organs, plant tissues or parts
of plants originate from the under source organism mentioned plant
families, preferably from the abovementioned plant genus, more
preferred from abovementioned plants spezies.
[0444] Transgenic plants produced in the process according to the
invention can be marketed directly. In the process according to the
invention, plants are understood as meaning all plant parts, plant
organs such as leaves, stalk, root, tubers or seeds or propagation
material or harvested material or the intact plant. In this
context, the seed encompasses all parts of the seed such as the
seed coats, epidermal cells, seed cells, endosperm or embryonic
tissue.
[0445] In yet another aspect, the invention also relates to
harvestable parts and to propagation material of the transgenic
plants according to the invention which either contain transgenic
plant cells expressing a nucleic acid molecule according to the
invention or which contains cells which show an reduced, decreased
or deleted cellular activity of the polypeptide of the invention,
e.g. a decreased expression level or lower activity of the
described protein.
[0446] Harvestable parts can be in principle any useful parts of a
plant, for example, flowers, pollen, seedlings, tubers, leaves,
stems, fruit, seeds, roots etc. Propagation material includes, for
example, seeds, fruits, cuttings, seedlings, tubers, rootstocks
etc. Preferred are seeds, seedlings, tubers or fruits as
harvestable or propagation material.
[0447] The invention furthermore relates to the use of the
transgenic plants according to the invention and of the cells, cell
cultures, parts--such as, for example, roots, leaves and the like
as mentioned above in the case of transgenic plant
organisms--derived from them, and to transgenic propagation
material such as seeds or fruits and the like as mentioned above,
for the production of foodstuffs or feeding stuffs, pharmaceuticals
or fine chemicals.
[0448] Accordingly in another embodiment, the present invention
relates to the use of the nucleic acid molecule in the plants,
plant cells or plant tissues, in a vector, or the polypeptide of
the present invention for increasing the growth of plants.
[0449] Another embodiment of the invention is a double-stranded RNA
molecule (dsRNA), whereby the sense strand of said double-stranded
RNA nucleic acid molecule has a homology of at least 30%, 35%, 40%,
45%, 50%, 55% or 60%, preferably 65%, 70%, 75% or 80%, more
preferably 85%, 90%, 95%, 96%, 97%, 98% or 99% to the nucleic acid
molecule of the invention or encoding a protein conferring the
expression of a protein having the biological activity of the
protein of the invention or comprising a fragment of at least 10
base paires (=bases, nt, nucleotides), preferably at least 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50,
especially preferably at least 40, 50, 60, 70, 80 or 90 base pairs,
very especially preferably at least 100, 200, 300 or 400 base
pairs, most preferably at least 500, 600, 700, 800, 900 or more
base pairs or at least 1000 or 2000 base pairs of a nucleic acid
molecule with a homology of at least 50%, 60%, 70%, 80% or 90%,
preferably 100% to a nucleic acid molecule conferring the
expression of a protein having the biological activity of the
protein of the invention or to the nucleic acid molecule of the
invention. In another preferred embodiment of the invention the
encoded sequence or its part-segment amounts to 17, 18, 19, 20, 21,
22, 23, 24, 25, 26 or 27 bases, preferably to 20, 21, 22, 23, 24 or
25 bases, whereby the homology of the sequence is similar to the
aforementioned homologies.
[0450] In a preferred embodiment of the invention the sense and
antisense strand of the double-stranded RNA are covalently bound or
are bound by weak chemical bonds such as hydrogen bonds to each
other and the antisense strand is essentially the complement of the
sense-RNA strand.
[0451] Yet another embodiment of the invention is an antisense
nucleic acid molecule, whereby the antisense nucleic acid molecule
has a homology of at least 30% to a nucleic acid molecule antisense
to a nucleic acid molecule encoding the protein encoded by the
nucleic acid molecule of the invention or encoding the protein of
the invention and conferring the expression of a protein having the
biological activity of a protein of the invention or an antisense
nucleic acid molecule comprising a fragment of at least 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or
50, especially preferably at least 60, 70, 80 or 90 base pairs,
very especially preferably at least 100, 200, 300 or 400 base
pairs, most preferably at least 500, 600, 700, 800, 900 or more
base pairs.
[0452] A further embodiment of the invention is a ribozyme, which
specifically cleaves a nucleic acid molecule conferring expression
of a protein having the biological activity of the protein of the
invention or a nucleic acid molecule encoding the protein encoded
by the nucleic acid molecule of the invention or the nucleic acid
molecule of the invention itself.
[0453] A further embodiment of the invention is an antibody, which
specifically binds to and therefore inhibits proteins encoded by
nucleic acid molecules conferring expression of a protein having
the biological activity of the protein of the invention or a
nucleic acid molecule encoding the protein encoded by the nucleic
acid molecule of the invention or the nucleic acid molecule of the
invention itself.
[0454] Yet another embodiments of the invention are a viral nucleic
acid molecule conferring the decline of a RNA molecule conferring
the expression of a protein having the biological activity of the
protein of the invention or of a nucleic acid molecule encoding the
protein encoded by the nucleic acid molecule of the invention or of
the nucleic acid molecule of the invention or a dominant-negative
mutant of the protein of the invention or a nucleic acid molecule
encoding such a dominant-negative mutant.
[0455] In one embodiment, the present invention relates to a method
for the identification of a gene product conferring an increase in
plant growth in a plant cell or a plant, comprising the following
steps: [0456] (a) contacting e.g. hybridising, the nucleic acid
molecules of a sample, e.g. plant cells, plant tissues, plants or
microorganisms or a nucleic acid library, which can contain a
candidate gene encoding a gene product conferring an increase in
plant growth after reduction or deletion of its expression, with
the nucleic acid molecule of the present invention; [0457] (b)
identifying the nucleic acid molecules, which hybridize under
relaxed stringent conditions with the nucleic acid molecule of the
present invention in particular to the nucleic acid molecule
sequence shown in SEQ ID NO: 1 or SEQ ID NO: 113 and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[0458] (c) reducing or deletion the expressing of the identified
nucleic acid molecules in the host cells; [0459] (d) assaying the
growth of plant cells or plants or plant parts; and [0460] (e)
identifying the nucleic acid molecule and its gene product which
reduction or deletion of expression confers an increase in growth
in the plant cells or plants or plant parts after expression
compared to the wild type.
[0461] Relaxed hybridisation conditions are: After standard
hybridisation procedures washing steps can be performed at low to
medium stringency conditions usually with washing conditions of
40.degree.-55.degree. C. and salt conditions between 2.times.SSC
and 0.2.times.SSC with 0.1% SDS in comparison to stringent washing
conditions as e.g. 60.degree. to 68.degree. C. with 0.1% SDS.
Further examples can be found in the references listed above for
the stringend hybridization conditions. Usually washing steps are
repeated with increasing stringency and length until a useful
signal to noise ratio is detected and depend on many factors as the
target, e.g. its purity, GC-content, size etc, the probe, e.g. its
length, is it a RNA or a DNA probe, salt conditions, washing or
hybridisation temperature, washing or hybridisation time etc.
[0462] In another embodiment, the present invention relates to a
method for the identification of a gene product the reduction of
which conferring an increased growth of plants, comprising the
following steps: [0463] (a) identifiying nucleic acid molecules of
an organism, which can contain a candidate gene encoding a gene
product conferring an increase in plant growth after reduction or
deletion of its expression, which are at least 20%, preferably 25%,
more preferably 30%, even more preferred are 35%. 40% or 50%, even
more preferred are 60%, 70% or 80%, most preferred are 90% or 95%
or more homolog to the nucleic acid molecule of the present
invention, for example via homology search in a data bank; [0464]
(b) reducing or deleting the expression of the identified nucleic
acid molecules in the host cells; [0465] (c) assaying the increase
in growth of the plant cells or plants or plant parts; and [0466]
(d) identifying the nucleic acid molecule and its gene product
which reduction or deletion of expression confers an increase in
plant growth compared to the wild type
[0467] The nucleic acid molecules identified can then be used for
the increase in plant growth in the same way as the nucleic acid
molecule of the present invention. Accordingly, in one embodiment,
the present invention relates to a process for the production of
plants with increased growth, comprising (a) identifying a nucleic
acid molecule according to aforementioned steps (a) to (d) and
harvesting the plants or plant parts transformed according to the
invention having an decreased cellular activity of a polypeptide
encoded by the isolated nucleic acid molecule compared to a wild
type.
[0468] Furthermore, in one embodiment, the present invention
relates to a method for the identification of a compound
stimulating the growth of said plant comprising: [0469] a)
contacting cells which express the polypeptide of the present
invention or its mRNA with a candidate compound under cell
cultivation conditions; [0470] b) assaying a reduction, decrease or
deletion in expression of said polypeptide or said mRNA; [0471] c)
comparing the expression level to a standard response made in the
absence of said candidate compound; whereby, a reduced, decreased
or deleted expression over the standard indicates that the compound
is stimulating the growth of the plant.
[0472] Furthermore, in one embodiment, the present invention
relates to a method for the screening for antagonists of the
activity of the polypeptide of the present invention, e.g. a
polypeptide conferring an increase in plant growth or a part
thereof after decreasing the cellular activity, e.g. of the
activity of a polypeptide having the biological activity
represented by the protein of the invention comprising: [0473] (a)
contacting plant cells, plant tissues or plants which express the
polypeptide according to the invention with a candidate compound or
a sample comprising a plurality of compounds under conditions which
permit the expression of the polypeptide of the present invention;
[0474] (b) assaying the growth of the plants or plant parts or the
polypeptide expression level in the plant cells, tissues or plants;
and [0475] (c) identifying an antagonist by comparing the measured
increase in plant growth or polypeptide expression level with a
standard growth rate or polypeptide expression level measured in
the absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level of plant growth
over the standard indicates that the compound or the sample
comprising said plurality of compounds is an antagonist.
[0476] Yet another embodiment of the invention relates to a process
for the identification of a compound conferring increased plant
growth comprising the following step: [0477] (a) culturing or
maintaining a plant or their tissues expressing the polypeptide of
the invention or a polynucleotide encoding said polypeptide and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with this readout system in the presence of a chemical compound or
a sample comprising a plurality of chemical compounds and capable
of providing a detectable signal in response to the binding of a
chemical compound to said polypeptide under conditions which permit
the depression of said readout system and of the protein of the
invention; and [0478] (b) identifying if the chemical compound is
an effective antagonist by detecting the presence or absence or
decrease or increase of a signal produced by said readout
system.
[0479] Said compound may be chemically synthesized or
microbiologically produced and/or comprised in, for example,
samples, e.g., cell extracts from, e.g., plants, animals or
microorganisms, e.g. pathogens. Furthermore, said compound(s) may
be known in the art but hitherto not known to be capable of
suppressing the polypeptide of the present invention. The reaction
mixture may be a cell free extract or may comprise a cell or tissue
culture. Suitable set ups for the method of the invention are known
to the person skilled in the art and are, for example, generally
described in Alberts et al., Molecular Biology of the Cell, third
edition (1994), in particular Chapter 17. The compounds may be,
e.g., added to the reaction mixture, culture medium, injected into
the cell or sprayed onto the plant.
[0480] If a sample containing a compound is identified in the
method of the invention, then it is either possible to isolate the
compound from the original sample identified as containing the
compound capable of activating or increasing the growth od plants
or part thereof, or one can further subdivide the original sample,
for example, if it consists of a plurality of different compounds,
so as to reduce the number of different substances per sample and
repeat the method with the subdivisions of the original sample.
Depending on the complexity of the samples, the steps described
above can be performed several times, preferably until the sample
identified according to the method of the invention only comprises
a limited number of or only one substance(s). Preferably said
sample comprises substances of similar chemical and/or physical
properties, and most preferably said substances are identical.
Preferably, the compound identified according to the described
method above or its derivative is further formulated in a form
suitable for the application in plant breeding or plant cell and
tissue culture.
[0481] The compounds which can be tested and identified according
to a method of the invention may be expression libraries, e.g.,
cDNA expression libraries, peptides, proteins, nucleic acids,
antibodies, small organic compounds, hormones, peptidomimetics,
PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp,
Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and
references cited supra). Said compounds can also be functional
derivatives or analogues of known inhibitors or activators. Methods
for the preparation of chemical derivatives and analogues are well
known to those skilled in the art and are described in, for
example, Beilstein, Handbook of Organic Chemistry, Springer edition
New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and
Organic Synthesis, Wiley, New York, USA. Furthermore, said
derivatives and analogues can be tested for their effects according
to methods known in the art. Furthermore, peptidomimetics and/or
computer aided design of appropriate derivatives and analogues can
be used, for example, according to the methods described above. The
cell or tissue that may be employed in the method of the invention
preferably is a host cell, plant cell or plant tissue of the
invention described in the embodiments hereinbefore.
[0482] Thus, in a further embodiment the invention relates to a
compound obtained or identified according to the method for
identifiying an antagonist of the invention said compound being an
antagonist of the polypeptide of the present invention.
[0483] Accordingly, in one embodiment, the present invention
further relates to a compound identified by the method for
identifying a compound of the present invention.
[0484] Said compound is, for example, a homolog of the polypeptide
of the present invention. Homologues of the polypeptid of the
present invention can be generated by mutagenesis, e.g., discrete
point mutation or truncation of the polypeptide of the present
invention. As used herein, the term "homologue" refers to a variant
form of the protein, which acts as an antagonist of the activity of
the polypeptide of the present invention. An anatgonist of said
protein has lost the biological activities of the polypeptide of
the present invention. In particular, said antagonist confers a
decrease of the expression level of the polypeptide of the present
invention and thereby the expression of said antagonist in a plant
or part thereof confers the increase of growth of the invention in
the plant or part thereof.
[0485] In one embodiment, the invention relates to an antibody
specifically recognizing the compound or antagonist of the present
invention.
[0486] The invention also relates to a diagnostic composition
comprising at least one of the aforementioned nucleic acid
molecules, vectors, proteins, antibodies or compounds of the
invention and optionally suitable means for detection.
[0487] The diagnostic composition of the present invention is
suitable for the isolation of mRNA from a cell and contacting the
mRNA so obtained with a probe comprising a nucleic acid probe as
described above under hybridizing conditions, detecting the
presence of mRNA hybridized to the probe, and thereby detecting the
expression of the protein in the cell. Further methods of detecting
the presence of a protein according to the present invention
comprise immunotechniques well known in the art, for example enzyme
linked immunoadsorbent assay. Furthermore, it is possible to use
the nucleic acid molecules according to the invention as molecular
markers or primers in plant breeding. Suitable means for detection
are well known to a person skilled in the art, e.g. buffers and
solutions for hydridization assays, e.g. the aforementioned
solutions and buffers, further and means for Southern-, Western-,
Northern-etc.--blots, as e.g. described in Sambrook et al. are
known.
[0488] In another embodiment, the present invention relates to a
kit comprising the nucleic acid molecule, the vector, the host
cell, the polypeptide, the antisense nucleic acid, the antibody,
plant cell, the plant or plant tissue, the harvestable part, the
propagation material and/or the compound and/or antagonist
identified according to the method of the invention.
[0489] The compounds of the kit of the present invention may be
packaged in containers such as vials, optionally with/in buffers
and/or solution. If appropriate, one or more of said components
might be packaged in one and the same container. Additionally or
alternatively, one or more of said components might be adsorbed to
a solid support as, e.g. a nitrocellulose filter, a glas plate, a
chip, or a nylon membrane or to the well of a micro titerplate. The
kit can be used for any of the herein described methods and
embodiments, e.g. for the production of the host cells, transgenic
plants, pharmaceutical compositions, detection of homologous
sequences, identification of antagonists or agonists, as food or
feed or as a supplement thereof or as supplement for the treating
of plants, etc.
[0490] Further, the kit can comprise instructions for the use of
the kit for any of said embodiments, in particular for the use for
producing plants or parst thereof having an increased growth
rate.
[0491] In one embodiment said kit comprises further a nucleic acid
molecule encoding one or more of the aforementioned protein, and/or
an antibody, a vector, a host cell, an antisense nucleic acid, a
plant cell or plant tissue or a plant.
[0492] In a further embodiment, the present invention relates to a
method for the production of a agricultural composition providing
the nucleic acid molecule, the vector or the polypeptide of the
invention or comprising the steps of the method according to the
invention for the identification of said compound or antagonist;
and formulating the nucleic acid molecule, the vector or the
polypeptide of the invention or the antagonist, or compound
identified according to the methods or processes of the present
invention or with use of the subject matters of the present
invention in a form applicable as plant agricultural
composition.
[0493] In another embodiment, the present invention relates to a
method for the production of plants with increased growth
supporting plant culture composition comprising the steps of the
method for of the present invention; and formulating the compound
identified in a form acceptable as agricultural composition.
[0494] Under "acceptable as agricultural composition" is
understood, that such a composition is in agreement with the laws
regulating the content of fungicides, plant nutrients, herbizides,
etc. Preferably such a composition is without any harm for the
protected plants and the animals (humans included) fed
therewith.
[0495] The present invention also pertains to several embodiments
relating to further uses and methods. The nucleic acid molecule,
polypeptide, protein homologues, fusion proteins, primers, vectors,
host cells, described herein can be used in one or more of the
following methods: identification of plants useful for the
production of plants with increased growth rate as mentioned and
related organisms; mapping of genomes; identification and
localization of sequences of interest; evolutionary studies;
determination of regions required for function; modulation of an
activity.
[0496] Accordingly, the nucleic acid molecules of the present
invention have a variety of uses. First, they may be used to
identify a plant or a close relative thereof. Also, they may be
used to identify the presence thereof or a relative thereof in a
mixed population of plants. By probing the extracted genomic DNA of
a culture of a unique or mixed population of plants under stringent
conditions with a probe spanning a region of the gene of the
present invention which is unique to this, one can ascertain
whether the present invention has been used or whether it or a
close relative is present.
[0497] Further, the nucleic acid molecule of the invention may be
sufficiently homologous to the sequences of related species such
that these nucleic acid molecules may serve as markers for the
construction of a genomic map in related organism or for
association mapping. Furthermore natural variation in the genomic
regions corresponding to nucleic acids of the invention or
homologous thereof may lead to variation in the activity of the
proteins of the invention and their homolgous and in consequence in
natural variation in the increase of the growth rate. In
consequence natural variation eventually also exists in form of
less active allelic variants leading already to a relative increase
in the growth rate. Different variants of the nucleic acids of the
invention, which correspond to different growth rate increasing
capabilities can be identified and used for marker assisted
breeding for plants showing increased growth rate.
[0498] Accordingly, the present invention relates to a method for
breeding plants with increased growth rate, comprising [0499] (a)
selecting a first plant variety capable of increased groth rate by
reducing, decreasing or deleting the expressing of the polypeptide
according to the invention; [0500] (b) associating the ability to
increase the growth rate with the expression level or the genomic
structure of the genes of the invention; [0501] (c) crossing the
first plant variety with a second plant variety, which
significantly differs in its ability to increase the growth rate;
and [0502] (d) identifying, which of the offspring varieties has
got the capacity to increase the growth rate by means of analyzing
the expression or genomic structure of the genes of the
invention.
[0503] The nucleic acid molecules of the invention are also useful
for evolutionary and protein structural studies. By comparing the
sequences of the invention to those encoding similar enzymes from
other organisms, the evolutionary relatedness of the organisms can
be assessed. Similarly, such a comparison permits an assessment of
which regions of the sequence are conserved and which are not,
which may aid in determining those regions of the protein which are
essential for the functioning of the enzyme. This type of
determination is of value for protein engineering studies and may
give an indication of what the protein can tolerate in terms of
mutagenesis without losing function.
[0504] Accordingly, the nucleic acid molecule of the invention can
be used for the identification of other nucleic acids conferring an
increase in growth rate after reduction, decrease or deletion of
their expression.
[0505] Further, the nucleic acid molecule of the invention or a
fragment of a gene conferring the expression of the polypeptide of
the invention, preferably comprising the nucleic acid molecule of
the invention, can be used for marker assisted breeding or
association maping of growth rate related traits.
[0506] Accordingly, the nucleic acid of the invention, the
polypeptide of the invention, the nucleic acid construct of the
invention, the plants, the host cell, the plant tissue, plant cell,
or the plant part thereof of the invention, the vector of the
invention, the antagonist identified with the method of the
invention, the nucleic acid molecule identified with the method of
the present invention, can be used for the production of plants
with increased growth rate.
[0507] These and other embodiments are disclosed and encompassed by
the description and examples of the present invention. Further
literature concerning any one of the methods, uses and compounds to
be employed in accordance with the present invention may be
retrieved from public libraries, using for example electronic
devices. For example the public database "Medline" may be utilized
which is available on the Internet, for example online at
ncbi.nlm.nih.gov/PubMed/medline.html. Further online databases and
addresses, such as ncbi.nlm.nih.gov/, infobiogen. fr/,
fmi.ch/biology/research-tools.html, tigr.org/, are known to the
person skilled in the art and can also be obtained using, e.g.,
lycos.com. An overview of patent information in biotechnology and a
survey of relevant sources of patent information useful for
retrospective searching and for current awareness is given in
Berks, TIBTECH 12 (1994), 352-364.
[0508] The present invention is illustrated by the examples, which
follow. The present examples illustrate the basic invention without
being intended as limiting the subject of the invention. The
content of all of the references, patent applications, patents and
published patent applications cited in the present patent
application is herewith incorporated by reference.
Example 1
Engineering of Arabidopsis Plants
[0509] A binary knock out vector was constructed based on the
modified pPZP binary vector backbone (comprising the kanamycin-gene
for bacterial selection; Hajdukiewicz, P. et al., 1994, Plant Mol.
Biol., 25: 989-994) and the selection marker bar-gene (De Block et
al., 1987, EMBO J. 6, 2513-2518) driven by the mas2'1' and mas271f
promoters (Velten et al., 1984, EMBO J. 3, 2723-2730; Mengiste,
Amedeo and Paszkowski, 1997, Plant J., 12, 945-948).
[0510] Examples of other usable binary vectors for insertional
mutagenesis are pBIN19, pBI101, pBinAR, pSun or pGPTV. An overview
over binary vectors and their specific features is given in Hellens
et al., 2000, Trends in plant Science, 5:446-451 and in Guerineau
F., Mullineaux P., 1993, Plant transformation and expression
vectors in plant molecular biology, LABFAX Series, (Croy R. R. D.,
ed.) pp. 121-127 Bios Scientific Publishers, Oxford.
Example 2
Plant Transformation and Analysis
[0511] The plasmid was transformed into Agrobacterium tumefaciens
(GV3101 pMP90; Koncz and Schell, 1986 Mol. Gen. Genet. 204:383-396)
using heat shock or electroporation protocols. Transformed colonies
were grown on YEB medium and selected by respective antibiotics
(Rif/Gent/Km) for 2 d at 28.degree. C. These agrobacteria cultures
were used for the plant transformation.
[0512] Arabidopsis thaliana of the ecotype C24 were grown and
transformed according to standard conditions (Bechtold, N., Ellis,
J., Pelletier, G. 1993. In planta Agrobacterium mediated gene
transfer by infiltration of Arabidopsis thaliana plants, C. R.
Acad. Sci. Paris 316:1194-1199; Bent, A. F., Clough, J. C., 1998;
Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana, PLANT J. 16:735-743).
[0513] Transformed plants (F1) were selected by the use of their
respective resistance marker. In case of BASTA.RTM.-resistance,
plantlets were sprayed four times at an interval of 2 to 3 days
with 0.02% BASTA.RTM. and transformed plants were allowed to set
seeds. 50-100 seedlings (F2) were subjected again to marker
selection, in case of BASTA-resistance by spaying with 0.1%
BASTA.RTM. on 4 consecutive days during the plantlet phase. Plants
segregating for a single resistance locus (approximately 3:1
resistant seedling to sensitive seedlings) were chosen for further
analysis. From these lines three of the resistant seedlings (F2)
were again allowed to set seeds and were tested for homozygosis
through in-vitro germination of their seeds (F3) on agar medium
containing the selection agent (BASTA.RTM., 15 mg/l ammonium
glufosinate, Pestanal, Riedel de Haen, Seelze, Germany). Those F2
lines which showed nearly 100% resistant offspring (F3) were
considered homozygote and taken for functional analysis.
[0514] For analysis, the plants were cultivated in a phytotron from
Swalof Weibull (Sweden) under the following conditions. After
stratification, the test plants were cultured in a 16 h light/8 h
dark rhythm at 20.degree. C., a humidity of 60% and a CO.sub.2
concentration of 400 ppm for 22-23 days. The light sources used
were Powerstar HQI-T 250 W/D Daylight lamps from Osram, which
generate light of a color spectrum similar to that of the sun with
a light intensity of 220 .mu.E/m.sup.2/s.sup.-1.
[0515] On days 24 after sowing, which correspond to approximately
day 17 after germination, in each case approximately 40 individual
plants of both the wild type (WT) and the KO lines which by visual
expection showed increased growth were studied. The fresh weight of
aboveground parts of transgenic lines and wild type (WT)
Arabidopsis plants was determined immediately thereafter, using a
precision balance. The differences between the results for the wild
type plants and the heaviest transgenic line were tested for
significance by means of an ANOVA for each line. One line KO 10488
with increased seed yield, leaf number and fresh weight could be
identified, see table 1a.
TABLE-US-00004 TABLE 1a Increase of biomass of transgenic KO10488
Arabidopsis plants in comparison to the MC24 wild type (means +/-
standard deviation). Seed Yield Leaf Number Fresh weight MC24 149.1
(+/- 37.9) 13.08 (+/- 7.9) 224.5 KO10488 189.8 (+/- 47.9) 13.6 (+/-
2) 237.5 For another line K014595 a strong increase in seed yield
and smaller increases in leaf number and fresh weight could be
identified.
TABLE-US-00005 TABLE 1b Increase of biomass especially seed yield
of transgenic KO14595 Arabidopsis plants in comparison to the MC24
wild type (means +/- standard deviation). Seed Yield Leaf Number
Fresh weight MC24 392.1 (+/- 98.6) 13.3 (+/- 6) 201.9 KO14595 557.1
(+/- 105.1) 13.5 (+/- 5) 208.1
Example 3
Molecular Analysis of KO Lines 10488 and 14595
[0516] Since the lines were preselected for single insertion loci
and a homozygous situation of the resistance marker, the disruption
(or mutation) of single genes through the integration of the T-DNA
were expected to have lead to the increased yield phenotype.
[0517] Genomic DNA was purified from approximately 100 mg of leaf
tissue from these lines using standard procedures (spin columns
from Qiagen, Hilden, Germany) The amplification of the insertion
side of the T-DNA KO line 10488 was achieved using an adaptor
PCR-method according to Spertini D, Beliveau C. and Bellemare G.,
1999, Biotechniques, 27, 308-314 using T-DNA specific primers
TABLE-US-00006 (SEQ ID NO: 96) LB1: 5'-TGA CGC CAT TTC GCC TTT
TCA-3' for the first and (SEQ ID NO: 97) LB2: 5'-CAG AAA TGG ATA
AAT AGC CTT GCT TCC-3' for the second PCR respectively.
[0518] For line KO014595 TAIL-PCR (Liu Y-G, Mitsukawa N, Oosumi T
and Whittier RF, 1995, Plant J. 8, 457-463 was preformed using the
degenerated primer ADP3 (5'-WGTGNAGWANCANAGA-3', SEQ ID: 253). As
primers specific for the T-DNA left border, in the first PCR round
primer LB1 (5'-TGA CGC CAT TTC GCC TTT TCA-3' SEQ ID: 262), for the
second round primer LB2 (5'-CAG AAA TGG ATA AAT AGC CTT GCT TCC-3'
SEQ ID 263) and for the last round primer LB3 (5'-CCA ATA CAT TAC
ACT AGC ATC TG-3'; SEQ ID: 264) was used.
[0519] Appropriate PCR-products were identified on agarose gels and
purified using columns and standard procedures (Qiagen, Hilden,
Germany). PCR-products were sequenced with additional
T-DNA-specific primers located towards the borders relative to the
primers used for amplification. For adaptor PCR products containing
left border sequences, primer LBseq (5'-CAA TAC ATT ACA CTA GCA TCT
G-3') (SEQ ID NO: 98) was used for sequencing reactions. The
resulting sequences were taken for comparison with the available
Arabidopsis genome sequence from Genbank using the blast algorithm
(Altschul et al., 1990. J Mol Biol, 215:403-410).
[0520] Details on PCR products used to identify the genomic locus
are given in table 2. Indicated are the identified annotated open
reading frame in the Arabidopsis genome, the estimated size of the
obtained PCR product (in base pairs), the T-DNA border (LB: left
border, RB: right border) for which the amplification was achieved,
the method which resulted in the indicated PCR product (explanation
see text above) and the respective restriction enzymes used in
adaptor PCR.
[0521] The identification of the insertion locus in each case was
confirmed by a control PCR, using one of the above mentioned
T-DNA-specific primers and a primer deduced from the identified
genomic locus, near to the insertion side. The amplification of a
PCR-product of the expected size from the insertion line using
these two primers proved the disruption of the identified locus by
the T-DNA integration.
TABLE-US-00007 TABLE 2 Details on PCR products used to identify the
down-regulated genomic locus in lines showing increased yield. SEQ
Restriction ID PCR- enzyme or NO ORF Product Border Method deg.
primer 1 at5g64410 620 bp LB Adaptor- Psp1406I/ PCR Bsp119I 113
at5g02270 550 bp LB TAIL- ADP3 PCR
[0522] Column 1 refers to the SEQ ID NO of the gene which has been
knocked out, column 2 refers to the genebank accession of the gene,
column 3 refers to the approximate length of the amplified PCR
product, column 4 refers to the T-DNA border for which the PCR
product was amplified, column 5 refers to the PCR method for
amplification and column 6 refers to restriction enzyme used in the
PCR method (for detailed explanation to columns 5 and 6 see text
above)
Example 4
Construction of Antisense Constructs for Repression of the Genes of
the Invention
[0523] A fragment of SEQ ID NO: 1 is amplified by PCR. To enable
cloning of the PCR product, restriction sites may be added to the
primers used for the amplification. Alternatively recombination
sites may be added to the primers to enable a recombination
reaction. The PCR fragment is either cloned or recombined into a
binary vector, preferently under control of a strong constitutive,
tissue or developmental specific promoter in a way, that the
orientation to the promoter is opposite to the direction of the
gene in its original genomic position.
[0524] The amplification of the fragment of the SEQ ID NO: 1 was
performed using the oligonucleotides that have been deduced from
the gene sequence:
TABLE-US-00008 at5g64410fw2: (SEQ ID NO: 94)
5'-atattaattaaGGTTCAAACATCATATCTTC-3' at5g64410rev2: (SEQ ID NO:
95) 5'-ataccatggCGGGTTTTGGGAAGCACCTTGG-3'
[0525] The Oligonucleotides have been solved in water to give a
concentration of 20 .mu.m. The PCR reaction contained 5 .mu.l
Herculase buffer (Stratagene), 0.4 .mu.l dNTPs (25 mM each)
(Amersham), 0.5 .mu.l Primer a07610fw, 0.5 .mu.l Primer a07610rev,
0.5 .mu.l Herculase (Stratagene), 0.5 .mu.l gDNA and 42.6 .mu.l
water. The PCR was performed on MJ-Cycler Tetrad (BioZym) with the
following program:
[0526] 4 mM 94.degree. C., followed by 30 cycles of 1 mM 94.degree.
C., 1 min 50.degree. C., 2 min 72.degree. C. followed by 10 min
72.degree. C. and cooling to 25.degree. C.
[0527] The PCR product has been purified using a Kit from Qiagen.
The DNA was subsequently digested with NcoI/PacI at 37.degree. C.
over night. The fragment was then cloned into the vector
1bxPcUbicolic--see FIG. 1--which has been digested with NcoI/PacI.
The resulting construct was named1bxPcUbianti at5g64410.
[0528] For Seq ID NO: 113, ORF at5g02270, die antisense repression
was carried out in a similar way using primer SEQ ID NO: 256 and
SEQ ID NO: 257 for the amplication of the gene fragment.
TABLE-US-00009 at5g02270fw2: (SEQ ID NO: 256) 5'
atattaattaaTGGAGCCGCATATGGTTAGG-3' at5g02270rev2: (SEQ ID NO: 257)
5'atccatggTCAGTCCGTGTTTCAAACTC-3'
Example 5
Construction of RNAi Constructs for Repression of the Genes of the
Invention
[0529] A fragment of SEQ ID NO: 1 is amplified by PCR. To enable
cloning of the PCR product, restriction sites may be added to the
primers used for the amplification. Alternatively recombination
sites may be added to the primers to enable a recombination
reaction. The PCR fragment is either cloned or recombined into a
binary vector, preferently under control of a strong constitutive,
tissue or developmental specific promoter in a way, that the
fragment is introduced twice in the vector as an inverted repeat,
the repeats separated by a DNA spacer.
[0530] The amplification of the fragment of the SEQ ID NO: 1 was
performed using the oligonucleotides that have been deduced from
the gene sequence:
TABLE-US-00010 at5g64410fw3: (SEQ ID NO: 99)
5'-ataggtaccGGTTCAAACATCATATCTTC-3' at5g64410rev3: (SEQ ID NO: 100)
5'-atagtcgacCGGGTTTTGGGAAGCACCTTGG-3'
[0531] The oligonucleotides have been solved in water to give a
concentration of 20 .mu.m. The PCR reaction contained 5 .mu.l
Herculase buffer (Stratagene), 0.4 .mu.l dNTPs (25 mM each)
(Amersham), 0.5 .mu.l Primer a07610fw, 0.5 .mu.l Primer a07610rev,
0.5 .mu.l Herculase (Stratagene), 0.5 .mu.l gDNA and 42.6 .mu.l
water. The PCR was performed on MJ-Cycler Tetrad (BioZym) with the
following programm:
[0532] 4 min 94.degree. C., followed by 30 cycles of 1 min
94.degree. C., 1 min 50.degree. C., 2 min 72.degree. C. followed by
10 min 72.degree. C. and cooling to 25.degree. C.
[0533] The PCR product has been purified using a Kit from Qiagen.
The DNA was subsequently digested with Asp718/SalI at 37.degree. C.
over night. The fragment was then cloned into the vector
10xPcUbispacer--see FIG. 2--which has been digested with
Asp718/SalI. The resulting construct was digested with XhoI/BsrGI
and the same Asp718/SalI digested PCR fragment was ligated into
this vector. Subsequently, the expression cassette giving rise to
BASTA resistance was ligated as XbaI fragment into this vector that
has been opened with XbaI and dephosphorilized before. The
resulting construct was named1bxPcUbiri3g07610.
[0534] For the RNAi repression of at5g02270, SEQ ID NO: 113 the
same procedure as described above was followed using the specific
primers:
TABLE-US-00011 at5g02270fw3: (SEQ ID NO: 260) 5'
ataggtaccTGGAGCCGCATATGGTTAGG-3' at5g02270rev3: (SEQ ID NO: 261)
5'atagtcgacTCAGTCCGTGTTTCAAACTC-3'
[0535] for the amplication of the gene specific fragment.
Example 6
Construction of Cosuppression Constructs for Repression of the
Genes of the Invention
[0536] A fragment of SEQ ID NO: 1 is amplified by PCR. To enable
cloning of the PCR product, restriction sites may be added to the
primers used for the amplification. Alternatively recombination
sites may be added to the primers to enable a recombination
reaction. The PCR fragment is either cloned or recombined into a
binary vector, preferently under control of a strong constitutive,
tissue or developmental specific promoter in a way, that the
orientation to the promoter is identical to the direction of the
gene in its original genomic position.
[0537] The amplification of the fragment of the SEQ ID NO: 1 was
performed using the oligonucleotides that have been deduced from
the gene sequence:
TABLE-US-00012 at5g64410w4: (SEQ ID NO: 101)
5'-ataccatggGGTTCAAACATCATATCTTC-3' at5g64410rev4: (SEQ ID NO: 102)
5'-atattaattaaCGGGTTTTGGGAAGCACCTTGG-3'
[0538] The oligonucleotides have been solved in water to give a
concentration of 20 .mu.m. The PCR reaction contained 5 .mu.l
Herculase buffer (Stratagene), 0.4 .mu.l dNTPs (25 mM each)
(Amersham), 0.5 .mu.l Primer a07610fw, 0.5 .mu.l Primer a07610rev,
0.5 .mu.l Herculase (Stratagene), 0.5 .mu.l gDNA and 42.6 .mu.l
water. The PCR was performed on MJ-Cycler Tetrad (BioZym) with the
following program:
[0539] 4 min 94.degree. C., followed by 30 cycles of 1 min
94.degree. C., 1 min 50.degree. C., 2 min 72.degree. C. followed by
10 min 72.degree. C. and cooling to 25.degree. C.
[0540] The PCR product has been purified using a Kit from Qiagen.
The DNA was subsequently digested with NcoI/PacI at 37.degree. C.
over night. The fragment was then cloned into the vector
1bxPcUbicolic--see FIG. 1--which has been digested with NcoI/PacI.
The resulting construct was named1bxPcUbicos3g07610.
[0541] For cosuppression of at5g02270, SEQ ID NO: 113 the same
procedure as described above was followed using the specific
primers:
TABLE-US-00013 at5g02270fw4: (SEQ ID NO: 258) 5'
atccatggTGGAGCCGCATATGGTTAGG-3' at5g02270rev4: (SEQ ID NO: 259) 5'
atattaattaaTCAGTCCGTGTTTCAAACTC-3'
[0542] for the amplication of the gene specific fragment.
Example 7
Reducing the Expression of the Genes of the Invention by Artificial
Transcription Factors
[0543] The genes of the invention and their homologous ORFS in
other species may also be down regulated by introducing a synthetic
specific repressor. For this purpose, a gene for a chimeric zinc
finger protein, which binds to a specific region in the regulatory
or coding region of the gene of interest or its homolog in other
spezies is constructed. The artificial zinc finger protein
comprises a specific DNA-binding domain consisting for example of
zinc finger and optionally a repression domain like the EAR domain
(Hiratsu et al., 2003. Plant J. 34(5), 733-739 Dominant repression
of target genes by chimeric repressors that include the EAR motif,
a repression domain, in Arabidopsis).
[0544] Expression of this chimeric repressor for example in plants
then results in specific repression of the target gene or of its
homologs in other plant species which leads to an increase in
yield. The experimental details especially about the design and
construction of specific zinc finger domains may be carried out as
described or as characterised in WO 01/52620 or Ordiz MI, (Proc.
Natl. Acad. Sci. USA, 2002, Vol. 99, Issue 20, 13290) or Guan,
(Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, Issue 20, 13296).
Example 8
Engineering Ryegrass Plants by Repressing the Nucleic Acid Sequence
Homologs of the Invention in Ryegrass
[0545] Seeds of several different ryegrass varieties can be used as
explant sources for transformation, including the commercial
variety Gunne available from Svalof Weibull Seed Company or the
variety Affinity. Seeds are surface-sterilized sequentially with 1%
Tween-20 for 1 minute, 100% bleach for 60 minutes, 3 rinses with 5
minutes each with de-ionized and distilled H.sub.2O, and then
germinated for 3-4 days on moist, sterile filter paper in the dark.
Seedlings are further sterilized for 1 minute with 1% Tween-20, 5
minutes with 75% bleach, and rinsed 3 times with ddH2O, 5 min
each.
[0546] Surface-sterilized seeds are placed on the callus induction
medium containing Murashige and Skoog basal salts and vitamins, 20
g/l sucrose, 150 mg/l asparagine, 500 mg/l casein hydrolysate, 3
g/l Phytagel, 10 mg/l BAP, and 5 mg/l dicamba. Plates are incubated
in the dark at 25.degree. C. for 4 weeks for seed germination and
embryogenic callus induction.
[0547] After 4 weeks on the callus induction medium, the shoots and
roots of the seedlings are trimmed away, the callus is transferred
to fresh media, is maintained in culture for another 4 weeks, and
is then transferred to MSO medium in light for 2 weeks. Several
pieces of callus (11-17 weeks old) are either strained through a 10
mesh sieve and put onto callus induction medium, or are cultured in
100 ml of liquid ryegrass callus induction media (same medium as
for callus induction with agar) in a 250 ml flask. The flask is
wrapped in foil and shaken at 175 rpm in the dark at 23.degree. C.
for 1 week. Sieving the liquid culture with a 40-mesh sieve is
collecting the cells. The fraction collected on the sieve is plated
and is cultured on solid ryegrass callus induction medium for 1
week in the dark at 25.degree. C. The callus is then transferred to
and is cultured on MS medium containing 1% sucrose for 2 weeks.
[0548] Transformation can be accomplished with either Agrobacterium
or with particle bombardment methods. An expression vector is
created containing a constitutive plant promoter and the repression
construct of the gene in a pUC vector. The plasmid DNA is prepared
from E. coli cells using with Qiagen kit according to
manufacturer's instruction. Approximately 2 g of embryogenic callus
is spread in the center of a sterile filter paper in a Petri dish.
An aliquot of liquid MSO with 10 g/l sucrose is added to the filter
paper. Gold particles (1.0 .mu.m in size) are coated with plasmid
DNA according to method of Sanford et al., 1993 and are delivered
to the embryogenic callus with the following parameters: 500 .mu.g
particles and 2 .mu.g DNA per shot, 1300 psi and a target distance
of 8.5 cm from stopping plate to plate of callus and 1 shot per
plate of callus.
[0549] After the bombardment, calli are transferred back to the
fresh callus development medium and maintained in the dark at room
temperature for a 1-week period. The callus is then transferred to
growth conditions in the light at 25.degree. C. to initiate embryo
differentiation with the appropriate selection agent, e.g. 250 nM
Arsenal, 5 mg/l PPT or 50 mg/l kanamycin. Shoots resistant to the
selection agent are appearing and once rooted are transferred to
soil.
[0550] Samples of the primary transgenic plants (T0) are analyzed
by PCR to confirm the presence of T-DNA. These results are
confirmed by Southern hybridization in which DNA is electrophoresed
on a 1% agarose gel and transferred to a positively charged nylon
membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit
(Roche Diagnostics) is used to prepare a digoxigenin-labelled probe
by PCR, and used as recommended by the manufacturer.
[0551] Transgenic T0 ryegrass plants are propagated vegetatively by
excising tillers. The transplanted tillers are maintained in the
greenhouse for 2 months until well established. The shoots are
defoliated and allowed to grow for 2 weeks.
Example 9
Engineering Soybean Plants by Repressing the Nucleic Acid Sequence
Homologs of the Invention in Soybean
[0552] Soybean can be transformed according to the following
modification of the method described in the Texas A&M U.S. Pat.
No. 5,164,310. Several commercial soybean varieties are amenable to
transformation by this method. The cultivar Jack (available from
the Illinois Seed Foundation) is commonly used for transformation.
Seeds are sterilized by immersion in 70% (v/v) ethanol for 6 min
and in 25% commercial bleach (NaOCl) supplemented with 0.1% (v/v)
Tween for 20 min, followed by rinsing 4 times with sterile double
distilled water. Removing the radicle, hypocotyl and one cotyledon
from each seedling propagates seven-day seedlings. Then, the
epicotyl with one cotyledon is transferred to fresh germination
media in petri dishes and incubated at 25.degree. C. under a 16-hr
photoperiod (approx. 100 .mu.E-m-2s-1) for three weeks. Axillary
nodes (approx. 4 mm in length) are cut from 3 to 4 week-old plants.
Axillary nodes are excised and incubated in Agrobacterium LBA4404
culture.
[0553] Many different binary vector systems have been described for
plant transformation (e.g. An, G. in Agrobacterium Protocols.
Methods in Molecular Biology vol 44, pp 47-62, Gartland K M A and M
R Davey eds. Humana Press, Totowa, N.J.). Many are based on the
vector pBIN19 described by Bevan (Nucleic Acid Research. 1984.
12:8711-8721) that includes a plant gene expression cassette
flanked by the left and right border sequences from the Ti plasmid
of Agrobacterium tumefaciens. A plant gene expression cassette
consists of at least two genes--a selection marker gene and a plant
promoter regulating the transcription of the repression cassette of
the trait gene. Various selection marker genes can be used as
described above, including the Arabidopsis gene encoding a mutated
acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos. 5,767,366
and 6,225,105). Similarly, various promoters can be used to
regulate the repression cassette to provide constitutive,
developmental, tissue or environmental repression of gene
transcription as described above. In this example, the 34S promoter
(GenBank Accession numbers M59930 and X16673) is used to provide
constitutive expression of the repression cassette, for example the
antisense, the RNAi or the cosuppression construct.
[0554] After the co-cultivation treatment, the explants are washed
and transferred to selection media supplemented with 500 mg/l
timentin. Shoots are excised and placed on a shoot elongation
medium. Shoots longer than 1 cm are placed on rooting medium for
two to four weeks prior to transplanting to soil.
[0555] The primary transgenic plants (T0) are analyzed by PCR to
confirm the presence of T-DNA. These results are confirmed by
Southern hybridization in which DNA is electrophoresed on a 1%
agarose gel and transferred to a positively charged nylon membrane
(Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche
Diagnostics) is used to prepare a digoxigenin-labelled probe by
PCR, and is used as recommended by the manufacturer.
Example 10
Engineering Corn Plants by Repressing Nucleic Acid Sequence
Homologs of the Invention in Corn
[0556] Transformation of maize (Zea Mays L.) is performed with a
modification of the method described by Ishida et al. (1996) Nature
Biotech 14745-50). Transformation is genotype-dependent in corn and
only specific genotypes are amenable to transformation and
regeneration. The inbred line A188 (University of Minnesota) or
hybrids with A188 as a parent are good sources of donor material
for transformation (Fromm et al., 1990, Biotech 8:833-839), but
other genotypes can be used successfully as well. Ears are
harvested from corn plants at approximately 11 days after
pollination (DAP) when the length of immature embryos is about 1 to
1.2 mm. Immature embryos are co-cultivated with Agrobacterium
tumefaciens that carry "super binary" vectors and transgenic plants
are recovered through organogenesis. The super binary vector system
of Japan Tobacco is described in WO94/00977 and WO95/06722. Vectors
can be constructed as described. Various selection marker genes can
be used including the maize gene encoding a mutated acetohydroxy
acid synthase (AHAS) enzyme (U.S. Pat. No. 6,025,541). Similarly,
various promoters can be used to regulate the repression cassette
to provide constitutive, developmental, tissue or environmental
repression of gene transcription. In this example, the 34S promoter
(GenBank Accession numbers M59930 and X16673) is used to provide
constitutive expression of the repression cassette.
[0557] Excised embryos are grown on callus induction medium, then
maize regeneration medium, containing imidazolinone as a selection
agent. The Petri plates are incubated in the light at 25.degree. C.
for 2 to 3 weeks, or until shoots develop. The green shoots are
transferred from each embryo to maize rooting medium and incubated
at 25.degree. C. for 2 to 3 weeks, until roots develop. The rooted
shoots are transplanted to soil in the greenhouse. T1 seeds are
produced from plants that exhibit tolerance to the imidazolinone
herbicides and which are PCR positive for the transgenes.
[0558] The T1 generation of single locus insertions of the T-DNA
can segregate for the transgene in a 3:1 ratio. Those progeny
containing one or two copies of the transgene are tolerant to the
imidazolinone herbicide. Homozygous T2 plants can exhibit similar
phenotypes as the T1 plants. Hybrid plants (F1 progeny) of
homozygous transgenic plants and non-transgenic plants can also
exhibited increased similar phenotypes.
Example 11
Engineering Wheat Plants by Repressing Nucleic Acid Sequence
Homologs of the Invention in Wheat
[0559] Transformation of wheat is performed with the method
described by Ishida et al. (1996 Nature Biotech. 14745-50). The
cultivar Bobwhite (available from CYMMIT, Mexico) is commonly used
in transformation. Immature embryos are co-cultivated with
Agrobacterium tumefaciens that carry "super binary" vectors, and
transgenic plants are recovered through organogenesis. The super
binary vector system of Japan Tobacco is described in WO94/00977
and WO95/06722. Vectors are constructed as described. Various
selection marker genes can be used including the maize gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S.
Pat. No. 6,025,541). Similarly, various promoters can be used to
regulate the repression cassette to provide constitutive,
developmental, tissue or environmental regulation of gene
repression. In this example, the 34S promoter (GenBank Accession
numbers M59930 and X16673) can be used to provide constitutive
expression of the repression cassette.
[0560] After incubation with Agrobacterium, the embryos are grown
on callus induction medium, then regeneration medium, containing
imidazolinone as a selection agent. The Petri plates are incubated
in the light at 25.degree. C. for 2 to 3 weeks until shoots
develop. The green shoots are transferred from each embryo to
rooting medium and incubated at 25.degree. C. for 2 to 3 weeks
until roots develop. The rooted shoots are transplanted to soil in
the greenhouse. T1 seeds are produced from plants that exhibit
tolerance to the imidazolinone herbicides and which are PCR
positive for the transgenes.
[0561] The T1 generation of single locus insertions of the T-DNA
can segregate for the transgene in a 3:1 ratio. Those progeny
containing one or two copies to the transgene are tolerant to the
imidazolinone herbicide. Homozygous T2 plants exhibit similar
phenotypes.
Example 12
Engineering Rapeseed/Canola Plants by Repressing Nucleic Acid
Sequence Homologs of the Invention in Rapeseed/Canola Plants
[0562] Cotyledonary petioles and hypocotyls of 5-6 day-old young
seedlings are used as explants for tissue culture and transformed
according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The
commercial cultivar Westar (Agriculture Canada) is the standard
variety used for transformation, but other varieties can also be
used.
[0563] Agrobacterium tumefaciens LBA4404 containing a binary vector
are used for canola transformation. Many different binary vector
systems have been described for plant transformation (e.g. An, G.
in Agrobacterium Protocols. Methods in Molecular Biology vol 44, pp
47-62, Gartland K M A and M R Davey eds. Humana Press, Totowa,
N.J.). Many are based on the vector pBIN19 described by Bevan
(Nucleic Acid Research. 1984. 12:8711-8721) that includes a plant
gene expression cassette flanked by the left and right border
sequences from the Ti plasmid of Agrobacterium tumefaciens. A plant
gene expression cassette consists of at least two genes--a
selection marker gene and a plant promoter regulating the
transcription of the repression cassette of the trait gene. Various
selection marker genes can be used including the Arabidopsis gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S.
Pat. Nos. 5,767,366 and 6,225,105). Similarly, various promoters
can be used to regulate the repression cassette to provide
constitutive, developmental, tissue or environmental regulation of
gene repression. In this example, the 34S promoter (GenBank
Accession numbers M59930 and X16673) can be used to provide
constitutive expression of the repression cassette.
[0564] Canola seeds are surface-sterilized in 70% ethanol for 2
min., and then in 30% Clorox with a drop of Tween-20 for 10 min,
followed by three rinses with sterilized distilled water. Seeds are
then germinated in vitro 5 days on half strength MS medium without
hormones, 1% sucrose, 0.7% Phytagar at 23.degree. C., 16 hours
light. The cotyledon petiole explants with the cotyledon attached
are excised from the in vitro seedlings, and are inoculated with
Agrobacterium by dipping the cut end of the petiole explant into
the bacterial suspension. The explants are then cultured for 2 days
on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar
at 23.degree. C., 16 hours light. After two days of co-cultivation
with Agrobacterium, the petiole explants are transferred to MSBAP-3
medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or
timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium
with cefotaxime, carbenicillin, or timentin and selection agent
until shoot regeneration. When the shoots are 5 to 10 mm in length,
they are cut and transferred to shoot elongation medium (MSBAP-0.5,
containing 0.5 mg/l BAP). Shoots of about 2 cm in length are
transferred to the rooting medium (MS0) for root induction.
[0565] Samples of the primary transgenic plants (T0) are analyzed
by PCR to confirm the presence of T-DNA. These results are
confirmed by Southern hybridization in which DNA is electrophoresed
on a 1 agarose gel and are transferred to a positively charged
nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit
(Roche Diagnostics) is used to prepare a digoxigenin-labelled probe
by PCR, and used as recommended by the manufacturer.
Example 13
Engineering Alfalfa Plants by Repressing Nucleic Acid Sequence
Homologs of the Invention in Alfalfa
[0566] A regenerating clone of alfalfa (Medicago sativa) is
transformed using the method of McKersie et al., 1999 Plant Physiol
119: 839-847. Regeneration and transformation of alfalfa is
genotype dependent and therefore a regenerating plant is required.
Methods to obtain regenerating plants have been described. For
example, these can be selected from the cultivar Rangelander
(Agriculture Canada) or any other commercial alfalfa variety as
described by Brown D C W and A Atanassov (1985. Plant Cell Tissue
Organ Culture 4: 111-112). Alternatively, the RA3 variety
(University of Wisconsin) has been selected for use in tissue
culture (Walker et al., 1978 Am J Bot 65:654-659).
[0567] Petiole explants are cocultivated with an overnight culture
of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999
Plant Physiol 119: 839-847) or LBA4404 containing a binary vector.
Many different binary vector systems have been described for plant
transformation (e.g. An, G. in Agrobacterium Protocols. Methods in
Molecular Biology vol 44, pp 47-62, Gartland K M A and M R Davey
eds. Humana Press, Totowa, N.J.). Many are based on the vector
pBIN19 described by Bevan (Nucleic Acid Research. 1984.
12:8711-8721) that includes a plant gene expression cassette
flanked by the left and right border sequences from the Ti plasmid
of Agrobacterium tumefaciens. A plant gene expression cassette
consists of at least two genes--a selection marker gene and a plant
promoter regulating the transcription of the repression cassette of
the trait gene. Various selection marker genes can be used
including the Arabidopsis gene encoding a mutated acetohydroxy acid
synthase (AHAS) enzyme (U.S. Pat. Nos. 5,767,366 and 6,225,105).
Similarly, various promoters can be used to regulate the repression
cassette that provides constitutive, developmental, tissue or
environmental regulation of gene repression. In this example, the
34S promoter (GenBank Accession numbers M59930 and X16673) can be
used to provide constitutive expression of the repression
cassette.
[0568] The explants are cocultivated for 3 d in the dark on SH
induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35
g/l K.sub.2SO.sub.4, and 100 .mu.m acetosyringinone. The explants
are washed in half-strength Murashige-Skoog medium (Murashige and
Skoog, 1962) and plated on the same SH induction medium without
acetosyringinone but with a suitable selection agent and suitable
antibiotic to inhibit Agrobacterium growth. After several weeks,
somatic embryos are transferred to BOi2Y development medium
containing no growth regulators, no antibiotics, and 50 g/l
sucrose. Somatic embryos are subsequently germinated on
half-strength Murashige-Skoog medium. Rooted seedlings are
transplanted into pots and grown in a greenhouse.
[0569] The T0 transgenic plants are propagated by node cuttings and
rooted in Turface growth medium. The plants are defoliated and
grown to a height of about 10 cm (approximately 2 weeks after
defoliation).
Example 14
Knock Out of the Genes of the Invention by Homologs
Recombination
[0570] Identifying mutations in the genes of the invention in
random mutagenized populations
a) In chemically or radiation mutated population [0571] Production
of chemically or radiation mutated populations is a common
technique and known to the skilled worker. Methods are described by
Koorneef et al. 1982 and the citations therein and by Lightner and
Caspar in "Methods in Molecular Biology" Vol 82. These techniques
usually induce pointmutations that can be identified in any known
gene using methods such as TILLING (McCallum et al., 2000) (Nat.
Biotech 18, 455-457); Till et al., (Methods Mol. Biol. 2003;
236:205-20) and Till et al., (BMC Plant Biol. 2004 Jul. 28; 4(1);
12). b) in T-DNA or transposon mutated population by reserve
genetics [0572] Reverse genetic strategies to identify insertion
mutants in genes of interest have been described for various cases
e.g. Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290); Sessions
et al., 2002 (Plant Cell 2002, 14, 2985-2994); Young et al., 2001,
(Plant Physiol. 2001, 125, 513-518); Koprek et al., 2000 (Plant J.
2000, 24, 253-263); Jeon et al., 2000 (Plant J. 2000, 22, 561-570);
Tissier et al., 1999 (Plant Cell 1999, 11, 1841-1852); Speulmann et
al., 1999 (Plant Cell 1999, 11, 1853-1866). Briefly material from
all plants of a large T-DNA or transposon mutagenized plant
population is harvested and genomic DNA prepared. Then the genomic
DNA is pooled following specific architectures as described for
example in Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290).
Pools of genomics DNAs are then screened by specific multiplex PCR
reactions detecting the combination of the insertional mutagen (eg
T-DNA or Transposon) and the gene of interest. Therefore PCR
reactions are run on the DNA pools with specific combinations of
T-DNA or transposon border primers and gene specific primers.
General rules for primer design can again be taken from Krysan et
al., 1999 (Plant Cell 1999, 11, 2283-2290) Rescreening of lower
levels DNA pools lead to the identifcation of individual plants in
which the gene of interest is disrupted by the insertional
mutagen.
Examples 15
Identification of Additional Homologs from Other Species, which can
be Knocked Out in a Similar Fashion in Order to Get Biomass
Increase
[0573] Homolgous genes in other species can be found with
techniques, well known to the person skilled in the art. For
example homologous genes can be found using low or medium
stringency hybridisation of cDNA or genomic libraries. The
construction and screening of libraries has extensively been
described for example by Sambrook, J. et al. (1989) "Molecular
Cloning: A Laboratory Manual". Cold Spring Harbor Laboratory Press
or Ausubel, F. M. et al. (1994) "Current Protocols in Molecular
Biology", John Wiley & Sons). Alternatively expression
libraries can be screened for homologous genes by antibodies
prepared against the orginial gene of interes.
[0574] If sequence information are available or can be produced,
homologous genes can be easily identified through standard database
searches with known algorithms like blastn, blastp or blastx. More
sophisticated bioinformatics programs like the Pedant-Pro Suite
from Biomax (Biomax Informatic AG, Matinsried, Germany) supports
the identification of homologs by homology searches but also by
functional categorisations. Identified homologous can then be knock
out in their source organisms in similar manners as described
above.
EQUIVALENTS
[0575] Those of ordinary skill in the art will recognize, or will
be able to ascertain using no more than routine experimentation,
many equivalents to the specific embodiments of the invention
described herein. Such equivalents are intended to be encompassed
by 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=US20110271402A1).
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=US20110271402A1).
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