MX2007003358A - Nucleic acid sequences encoding proteins associated with abiotic stress response and plant cells and plants with increased tolerance to environmental stress - Google Patents
Nucleic acid sequences encoding proteins associated with abiotic stress response and plant cells and plants with increased tolerance to environmental stressInfo
- Publication number
- MX2007003358A MX2007003358A MXMX/A/2007/003358A MX2007003358A MX2007003358A MX 2007003358 A MX2007003358 A MX 2007003358A MX 2007003358 A MX2007003358 A MX 2007003358A MX 2007003358 A MX2007003358 A MX 2007003358A
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- Prior art keywords
- nucleic acid
- acid molecule
- tolerance
- plant
- plant cell
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Abstract
This invention relates generally to nucleic acid sequences encoding proteins that are associated with abiotic stress responses and abiotic stress tolerance in plants. In particular, this invention relates to nucleic acid sequences encoding proteins that confer drought, heat, cold, and/or salt tolerance to plants.
Description
NUCLEIC ACID SEQUENCES THAT CODIFY ASSOCIATED PROTEINS WITH RESPONSE TO ABIDICAL AGGRESSION AND CELLS OF
PLANTS AND PLANTS WITH TOLERANCE INCREASED TO AGGRESSION
ENVIRONMENTAL
This invention relates generally to nucleic acid sequences encoding proteins that are associated with responses to abiotic aggression and tolerance to abiotic aggression in plants. In particular, this invention relates to nucleic acid sequences that encode proteins that confer plants tolerance to drought, heat, cold and / or salt. In particular, this invention relates to nucleic acid sequences that encode proteins that confer on plants tolerance and / or resistance to drought, heat, cold and / or salt, preferably altering the metabolic activity that leads plants to tolerance and / or resistance to drought, heat, cold and / or salt. The invention also deals with methods for producing, selecting and reproducing such plant or plant cells and methods for detecting stress in plant or plant cells. Abiotic environmental aggressions such as drought aggression, salinity aggression, heat aggression and cold aggression are important factors limiting the growth and productivity of plants (Boyer,
1982. Science 218, 443-448). Crop losses and crop yield losses from major crops such as rice, corn (cob) and wheat caused by these aggressions represent a significant economic and political factor and contribute to food deficiencies in many underdeveloped countries. Plants are normally exposed during their life cycle to conditions of reduced environmental water content. Most plants have developed strategies to protect against these conditions of minimum water level or desiccation (drought). However, if the severity and duration of the drought conditions are very large, the effects on the development, growth and production of plants of most forage plants are profound. Continuous exposure to drought causes greater alterations in the metabolism of the plant. These large changes in metabolism ultimately lead to cell death and consequently production losses. Developing plants that are tolerant to aggression is a strategy that has the potential to solve or mediate at least some of these problems (McKersie and Leshem, 1994. Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers). However, strategies of reproduction of traditional plants to develop new lines of plants that exhibit resistance (tolerance) to
These types of aggressions are relatively slow and require specific resistant lines to cross with the desired line. Limited germplasm resources for tolerance and incompatibility to stress in crosses between remotely related plant species represent significant problems encountered in conventional breeding. In addition, the cellular processes that lead to tolerance to drought, cold and salt are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic trajectories (McKersie and Leshem, 1994. Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers ). This multi-component nature of tolerance to aggression has not only made breeding for largely unsuccessful tolerance, but has also limited the ability to genetically engineer plants that are tolerant to aggression using biotechnological methods. Aggressions due to drought, heat, cold and salt have an important common theme for plant growth and that there is water availability. The plants are exposed during their complete life cycle to conditions of reduced environmental water content. Most plants have developed strategies to protect against these conditions. However, if the severity and duration of the drought conditions are very large, the effects on the
The development, growth and yield of plants of most forage plants are deep. Since the elevated saline content in some lands results in less water available for cellular incorporation, its effect is similar to those observed under drought conditions. In addition, under freezing temperatures, water free of plant cells as a result of ice formation that starts in the apoplast and removes water from the symplast (McKersie and Leshem, 1994, Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers). Commonly, the molecular response mechanisms of a plant to each of these stress conditions are similar. Current research results indicate that tolerance to drought is a complex quantitative trait and that no real diagnostic marker is yet available. High concentrations of salt or dehydration can cause damage at the cellular level during drought aggression, but the precise damage is not completely clear (Bray, 1997. Trends Plant Sci. 2, 48-54). This lack of mechanistic understanding makes it difficult to design a transgenic method to improve drought tolerance. However, an important consequence of the damage can be the production of oxygen reactive radicals that cause cellular damage, such lipid peroxidation or modification of proteins or nucleic acid. Details of radical chemistry have been described
free to oxygen and its reaction with cellular components such as cell membranes (McKersie and Leshem, 1994, Stress and Stress Coping in Cultivated Plants, Kluwer Academic Publishers). There is a need to identify genes expressed in aggression tolerant plants that have the ability to confer stress resistance to their host plant and other plant species. It is an object of this invention to identify new methods for detecting or conferring tolerance and / or stress resistance in plants or plant cells. It is the object of this invention to identify new unique genes capable of conferring tolerance to aggression on plants in expression or overexpression. It is a further object of this invention to identify, produce and reproduce new, unique, tolerant and / or stress resistant plant or plant cells and methods for inducing and detecting tolerance and / or stress resistance in plants or plant cells. It is an additional object to identify new methods to detect tolerance and / or resistance to stress in plants or plant cells. This invention fulfills in part the need to identify new unique genes, capable of conferring stress tolerance to plants in the expression or over-
expression of endogenous and / or exogenous genes. The present invention provides genes from useful plants. These genes are encoded for stress related proteins (SRP) capable of conferring increased tolerance to environmental aggression when compared to a wild-type variety of the plant cell or plants in overexpression. The present invention also provides methods for modifying stress tolerance of a plant, comprising, modifying the expression of a SRP (stress-related protein) nucleic acid, wherein the SRP is as described below. The invention holds that this method can be carried out in such a way that stress tolerance increases or decreases. Preferably, tolerance to stress in a plant is increased through increased expression of an SRP nucleic acid. Increased expression of the endogenous gene encoding SRP can be achieved, for example, by increasing the resistance of the promoter used to drive the transcription of the gene and / or increasing the number of copies of the gene and its regulatory elements. Strong gene expression and multiple copies of the gene lead to increased levels of mRNA and target protein. Current methods for over-expressing proteins involve the cloning of the gene of interest and place it, in a construction, after a
suitable promoter / polyadenylation signal, and splice site, and introducing the construct into an appropriate host cell. The invention is also directed to methods for over-expressing an endogenous gene in a cell, comprising introducing a vector containing a transcriptional regulatory sequence and one or more amplifiable markers within the cell, allowing the vector to integrate into the cell's genome by non-homologous recombination, and allowing the over-expression of the endogenous gene in the cell. The invention is also directed to methods for overexpression of an exogenous gene in a cell, comprising introducing a vector containing a transcriptional regulatory sequence and one or more amplifiable markers within the cell, allowing the vector to integrate into the cell genome by non-homologous recombination, and allowing over-expression of the exogenous gene in the cell. In a preferred embodiment the expression or overexpression of an SRP nucleic acid from Zea mays confers tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in a cell / plant of Glycine max and vice versa, or an SRP nucleic acid from Zea mays confers tolerance and / or increased resistance to environmental aggression when
compares with a corresponding non-transformed wild-type plant cell in a cell / plant of Glycine max, Brassica napus or Oryza sativa and vice versa, or an SRP nucleic acid from Glycine max confers tolerance and / or increased resistance to environmental aggression when compares with a corresponding non-transformed wild type plant cell in a cell / plant of Zea mays, Brassica napus or Oryza sativa and vice versa, or an SRP nucleic acid from Brassica napus confers tolerance and / or increased resistance to environmental aggression when compares with a corresponding non-transformed wild type plant cell in a cell / plant of Glycine max, Zea mays or Oryza sativa and vice versa, or a SRP nucleic acid from Oryza sativa confers tolerance and / or increased resistance to environmental aggression when compare with a corresponding non-transformed wild-type plant cell in a cell / plant of Glycine max, Brassica napus or Zea mays and vice versa. The present invention provides a transgenic plant cell, wherein the expression of the nucleic acid sequence in the plant cell results in tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell. . The present invention provides a cell
transgenic plant transformed by nucleic acid encoding the Stress Related Protein (SRP), selected from the group consisting of: a) a nucleic acid molecule encoding one of the polypeptides according to SEQ. FROM IDENT. DO NOT. : 2,
6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62,
66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118,
122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166,
170 r 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214 and / or
218 r and / or 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,
242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,
266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,
290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,
314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336,
338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,
362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,
386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,
410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432,
434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456,
458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480,
482 r 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504,
506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528,
530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552,
554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576,
578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600,
602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624,
626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648,
650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672,
674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696,
698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720,
722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744,
746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768,
770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792,
794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816,
818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840,
842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864,
866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888,
890, 892, 894, 896, 898, 900, 902, | 904, 906, 908, 910, 912,
914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936,
938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960,
962, 964, 966, 968, 970, 972 and / or 974 or a fragment thereof, which confers an increased tolerance and / or resistance to environmental aggression in an organism or a part thereof; b) a nucleic acid molecule comprising one of the nucleic acid molecule according to SEQ. FROM IDENT. DO NOT. : 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97 , 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157,
161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205,
209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237,
239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,
287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309,
311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,
337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361,
363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413,
415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439,
441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463,
465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489,
491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539,
541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,
565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587,
589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611,
613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659,
661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,
685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707,
709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731,
733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779,
781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803,
805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827,
829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851,
853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875,
877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899,
901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923,
925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947,
949, 951, 953, 955, 957, 959, 961,? 963, 965, 967, 969, 971 and / or
973; c) a nucleic acid molecule whose sequence can be deduced from a polypeptide sequence encoded by a nucleic acid molecule of (a) or (b) as a result of the degeneracy of the genetic code and which confers an increased tolerance and / or resistance to the environmental aggression when compared to a corresponding non-transformed wild-type plant in an organism or a part of it; d) a nucleic acid molecule which encodes a polypeptide which has at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and conferring a tolerance and / or increased resistance to environmental aggression in an organism or a part thereof; e) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under
severe hybridization conditions and conferring a tolerance and / or increased resistance to environmental aggression in an organism or a part thereof; f) a nucleic acid molecule which comprises a nucleic acid molecule which is obtained by amplifying nucleic acid molecules from a cDNA library or a genomic library using the primers as shown in table 2 and conferring a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant in an organism or a part thereof; g) a nucleic acid molecule encoding a polypeptide which is isolated with the aid of monoclonal antibodies against a polypeptide encoded by one of the nucleic acid molecules of (a) to (f) and conferring increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant in an organism or a part thereof; and h) a nucleic acid molecule which is obtainable by selecting a suitable nucleic acid library under severe hybridization conditions with a probe comprising one of the sequences of the
nucleic acid from (a) to (g) 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 (k) and conferring an increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant in an organism or a part thereof. or which comprises a sequence which is complementary to it. For the purpose of the present invention, SEC.
FROM IDENT. NOS .: and the expression: SEC. FROM IDENT. NO .: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69,
73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121,
125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169,
173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or
217; and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,
241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,
265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,
287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,
309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331,
333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,
357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,
379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399,
401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423,
425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447,
449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469,
471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,
495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,
517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539,
541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,
565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585,
587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607,
609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631,
633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655,
657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677,
679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699,
701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721,
723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745,
747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,
769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789,
791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813,
815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835,
837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857,
859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879,
881, 883, 885, 887, 889, 891, 893. 895, 897, 899, 901,
903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925,
927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947,
949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973
they are summarized and named as "SECTION OF IDENTITY NO .: (4n + l) for n = 0 to 54 and (2n +1) for n = 110 to 487". This means, throughout the present specification, the term: "SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29,
33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217; and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,
241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573,
575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595,
597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617,
619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639,
641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661,
663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683,
685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705,
707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727,
729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749,
751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771,
773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793,
795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815,
817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837,
839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859,
861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881,
883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903,
905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925,
927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947,
949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973"and the term" SEC. FROM IDENT. DO NOT. : (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487"are identical and interchangeably in the present context For the purpose of the present invention, SEQ ID NOS. : and the expression: SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74 , 78, 82, 86, 90, 94, 98, 102, 106, 110, 114,
118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158,
162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202,
206, 210, 214 and / or 218 and / or 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,
264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,
288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,
312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334,
336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,
380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400,
402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422,
424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444,
446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488,
490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510,
512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532,
534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554,
556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598,
600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620,
622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642,
644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664,
666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708,
710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972 and / or 974 are summarized and referred to as "SEQ ID NO:: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487". This means, throughout the present specification, the term: SEC. FROM IDENT. DO NOT. : 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98 , 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198 , 202, 206, 210, 214 and / or 218 and / or 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256 , 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,
288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,
312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334,
336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,
360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402,
404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424,
426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446,
448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468,
470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512,
514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534,
536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556,
558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578,
580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622,
624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644,
646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668,
670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692,
694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740,
742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762,
764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784,
786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806,
808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852,
854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 96 ?, 962, 964, 966, 968, 970, 972 and / or 974"and the term" SEC. FROM IDENT. DO NOT. : (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487"are identical and interchangeably in the present context The invention provides that the environmental aggression may be by salinity, drought, temperature, metals, pathogenic and oxidative aggressions, or combinations thereof In the cell of the transgenic plant of the invention, the expression of the nucleic acid results in increased tolerance to an environmental aggression when compared to a corresponding non-transformed wild-type plant cell. Here, the environmental aggression is selected from the group consisting of salinity, drought, temperature, metals, chemicals, pathogenic and oxidative aggressions or combinations of them, the terms "increased", "elevated",
"extended", "enhanced", "enhanced" or "amplified" are related to a corresponding change of a property in an organism, a part of an organism such as a tissue.
seed, root, leaf, flower, etc., or in a cell and are interchangeable. Preferably, the total activity in the volume increases or improves in cases if the increase or improvement is related to the increase or improvement of an activity of a genetic product, regardless of whether the amount of the genetic product or the specific activity of the genetic product or both is increased or improved or if the amount, stability or translation efficiency of the nucleic acid sequence or genetic coding for the gene product is increased or improved. The terms "reduction", "decrease" or "elimination" are related to a corresponding change of a property in an organism, a part of an organism such as a tissue, seed, root, leaf, flower, etc., or in a cell. Preferably, the total activity in the volume is reduced, reduced or eliminated in cases if the reduction, decrease or elimination is related to the reduction, decrease or elimination of an activity of a genetic product, regardless of whether the amount of the genetic product or the specific activity of the gene product or both is reduced, decreased or eliminated or if the amount, stability or translation efficiency of the nucleic acid sequence or genetic coding for the gene product is reduced, decreased or eliminated. The terms "increase" or "decrease" are related to a corresponding change of a property in a
organism or in a part of an organism, such as a tissue, seed, root, leaf, flower, etc., or in a cell. Preferably, the total activity in the volume increases in cases in which the increase is related to the increase of an activity of a genetic product, independent of whether the amount of the genetic product or the specific activity of the genetic product or both is increased or is generated or if the amount, stability or translation efficiency of the nucleic acid sequence or genetic coding for the gene product is increased. Under "change of a property" it is understood that the activity, expression level or quantity of a genetic product or the metabolic content is changed in a specific volume in relation to a corresponding volume of a control, reference or wild type, including the creation de novo of the activity or expression. The terms "increase" or "decrease" include the change of ownership in only parts of the subject of the present invention, for example, the modification may be in compartment of a cell, such as an organelle, or in part of a plant, as tissue, seed, root, leaf, flower, etc., but it is not detectable if the total subject, that is, the whole cell or plant is tested. Preferably, the increase or decrease is found at the cellular level, thus the term "increase of one
activity "or" increase of a metabolic content "is related to the cellular increase compared to the wild-type cell.Therefore, the term" increase "or" decrease "means that the specific activity of an enzyme as well as the amount of a compound or metabolite, for example, of a polypeptide, a nucleic acid molecule or of the fine chemical of the invention or a coding mRNA or DNA, can be increased or decreased in one volume.The terms "wild type", "control" or "reference "are interchangeable and may be a cell or a part of organisms such as an organelle or a tissue, or an organism, in particular a microorganism or a plant, which is not modified or treated in accordance with the process described herein. According to the invention, therefore, the cell or a separate one of the organisms such as an organelle or a tissue, or an organism, in particular a microorganism or a plant used as a wild type. stre, 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. In this way, the wild type, control or reference is treated identically or as identically as possible, arguing that only conditions
or properties could be different which does not influence the amount of the property tested. Preferably, any comparison is carried out under analogous conditions. The term "analogous conditions" means that all conditions such as, for example, growing or growing conditions, test conditions (such as buffer composition, temperature, substrates, pathogen strain, concentrations and the like) remain identical among the experiments that are compared. The "reference", "control", or "wild type" is preferably a subject, for example, an organelle, a cell, a tissue, an organism, in particular a plant or a microorganism, which was not modified or treated according to the process described in the present 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 metabome as similar as possible to the subject of the present invention. Preferably, the term organelle, cell, tissue or organism, in particular plant or microorganism of "reference", "control" or "wild type", relates to an organelle, cell, tissue or organism, in particular plant or microorganism, which is almost genetically identical to the organelle, cell, tissue or organism, in particular a microorganism 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. More preferably, "reference", "control" or "wild type" is a subject, for example an organelle, a cell, a tissue, an organism, which is genetically identical to the organism, cell or organelle used in accordance with the process of the invention except that the nucleic acid molecules responsible for or conferring activity or the genetic product encoded by them are modified, manipulated, exchanged or introduced according to the inventive process. Preferably, the reference, control or wild type differ from the subject of the present invention only in the cellular activity of the polypeptide of the invention, for example, as a result of an increase in the level of the nucleic acid molecule of the present invention or an increase in the specific activity of the polypeptide of the invention, for example, by, or at the level of expression or activity of a protein having the activity of a Stress Related Protein (SRP) or its homologs, its biochemical causes or genetic and tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant.
In this case, a control, reference or wild type that differ from the subject of the present invention only by not being subjected to the process of the invention can not be provided, a control, reference or wild type can be an organism in which the cause for the modulation of an activity that confers tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant or expression of the nucleic acid molecule of the invention as described herein has been restored or switched off, for example, by inactivating the expression of the gene product responsible, for example, by inhibiting anti-sense, by inactivating an activator or agonist, by activating an inhibitor or antagonist, by inhibiting the addition of inhibitory antibodies, by adding compounds active substances such as hormones, introducing negative dominant mutants, etc. A genetic production can, for example, be inactivated by introducing inactivation point mutations, which lead to an inhibition of enzymatic activity or a destabilization or an inhibition of the ability to bind to cofactors, etc. Accordingly, the preferred reference subject is the starting subject of the present process of the invention. Preferably, the reference and subject matter of the invention are compared after standardization and
normalization, for example, to the amount of RNA, total DNA or protein or activity or expression of reference genes, such as prior genes, such as ubiquitin, actin or ribosomal proteins. There are a number of mechanisms through which a modification of the protein, for example, the polypeptide of the invention can directly or indirectly affect the yield, production and / or production efficiency of the amino acid. For example, the number of the molecule or specific activity of the polypeptide or the nucleic acid molecule can be increased. Larger amounts of the fine chemical can be produced if the polypeptide or the nucleic acid of the invention is expressed de novo in an organism lacking the activity of the protein. However, it is also possible to increase the expression of the gene which naturally occurs in organisms, for example by modifying the regulation of the gene, or by increasing the stability of the corresponding mRNA or the corresponding gene product encoded by the nucleic acid molecule of the gene. the invention, or by introducing homologous genes from other organisms which are regulated differently, for example, without sensitive feedback. This also applies analogously to the combined increased expression of the nucleic acid molecule of the
present invention or its genetic product with that of the additional enzymes of the amino acid biosynthesis paths, for example, which are useful for the synthesis of fine chemicals. The increase, decrease or modulation according to this invention can be constitutive, for example, due to a stable permanent transgenic expression or 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 behavior of a gene that confers the expression of the polypeptide of the invention or transient, for example, due to a transient transformation or temporary addition of a modulator such as an agonist or antagonist or inducible, for example, after transformation with a inducible construct carrying the nucleic acid molecule of the invention under the control of an inducible promoter and adding the inducer, for example, tetracycline or as described hereinafter. The increase in the activity of the polypeptide amounts in a cell, a tissue, an organelle, an organ or an organism or a part thereof preferably at least 5%, preferably at least 20%, or at least 50%, especially preferably at least 70%, 80%, 90% or more, most preferably at least 200%, more
preferably they are at least 500% or more in comparison to the control, reference or wild type. 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 expression of a protein in question in a cell, for example, a plant cell or a microorganism and the detection of an increase in the level of fine chemical in comparison to a control is an easy test and can be carried out as described in the state of the art. The term "increase" includes, that a compound or activity is introduced into a de novo cell or that the compound or activity has not been detectable before, in other words it is "generated". Therefore, in the following, the term "increase" also includes the term "generate" or "stimulate". The increased activity is manifested by an increase in the fine chemical. The cells of transformed plants are compared to the corresponding non-transformed wild type of the same genus and species under other identical conditions (such as, for example, culture conditions, age of the plants and the like). In this context, an increase in tolerance and / or resistance to environmental aggression of at least 10%,advantageously at least 20%, preferably at least 30%, especially preferably at least 40%, 50%, or 60%, most especially preferably at least 70%, 80%, 90%, 95% or even 100% or more, in comparison with the non-transformed organism it is advantageous. The present invention provides a transgenic plant cell, wherein the expression of the nucleic acid sequence in the plant cell results in tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell. A preferred wild-type plant cell is an untransformed Arabidopsis plant cell, an example here is the wild-type C24 Arabidopsis (Nottingham Arabidopsis Stock Center, UK, NASC Stock N906). transformed plants selected from the group consisting of corn, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, flax, borage, safflower, linseed, primrose , rapeseed, wild turnip, marigold, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, peas, alfalfa, coffee, cocoa, tea, Salix species, oil palm, coconut, perennial herbs and fodder.
Plant cells most preferred wildtype are a plant cell Linum untransformed, preferably Linum usitatissimum, more preferably the variety Brigitta, Golda, Gold Merchant, Helle, Juliel, Olpina, livia, Marlin, Maedgold, Sporpion, Serenade, Linus, Taunus, Lifax or Liviola, a plant cell Heliantus untransformed preferably Helianthus annuus, more preferably the variety Aurasol, Capella, Flavia, Flores, Jazzy, Palulo, Pegasol, PIR64A54, Rigasol, Sariuca, sidereal, Sunny, Alenka, Candisol or Floyd, or a plant cell untransformed Brassica, preferably Brassica napus, more preferably the variety Dorothy, avoids, Heros, Hyola, Kimbar, Lambada, Licolly, Liconira, Licosmos, Lisonne, Mistral, Passat, Serator, Siapula, Sponsor, Star , Daviar, Hybridol, Baical, Olga, Lara, Doublol, Karola, Falcon, Spirit, Olymp, Zeus, Libero, Kyola, Licord, Lion, Lirajet, Lisbeth, Magnum, Maja, Mendel, Mica, Mohican, Olpop, Ontarion, Panthar , Prinoe, Pronio, Susanna, Talani, Titan, Transfer, Wiking, Woltan, Zeniah, Artus, Contact or Smart. Expression of the nucleic acid sequence in the plant cell can directly or indirectly influence the tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild type plant of the cells of transformed plants.
Preferably, they include the activity of the above metabolites. Preferably, the tolerance and / or increased resistance to environmental aggression when compared to a corresponding wild-type non-transformed plant can be altered by transformation with one or more nucleic acid encoding the Stress Related Protein (SRP) selected from the group comprising the nucleic acid according to SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 or homologs of the sequences mentioned above. It is within the scope of the invention to identify the genes encoded by a nucleic acid sequence selected from the group consisting of the nucleic acid according to SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs thereof in target plants, especially forage plants and then express the corresponding gene to achieve tolerance and / or increased resistance to environmental aggression. Consequently, the invention is not limited to a specific plant. A protein having an activity that confers an increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant preferably has the
structure of the polypeptide described herein, of the polypeptide according to SEQ. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487 or functional homologs thereof as described herein, or is encoded by the characterized nucleic acid molecule in the present or the nucleic acid molecule according to the invention, for example, by the nucleic acid molecule according to SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 or their functional homologs described herein and having activity mentioned herein. It is also within the scope of the invention to select plant or plant cells for tolerance and / or increased resistance to environmental aggression by selecting plant cells under stress conditions for tolerance and / or increased resistance to environmental aggression when compared to Stress-free conditions This allows the selection of plants with tolerance and / or increased resistance to environmental aggression without the identification of genes or visual symptoms. With the invention, it is also possible to reproduce cells of plants or plants towards tolerance and / or increased resistance to environmental aggression by selecting plant cells under stress conditions for tolerance and / or increased resistance to. environmental aggression when compared to stress-free conditions and selecting those
with tolerance and / or increased resistance to environmental aggression. The selection for tolerance and / or increased resistance to environmental aggression is faster and easier than for example, the selection of genes. The selection is well known to those skilled in the art and generally refers to the search for a particular attribute or feature. In the invention, this feature in a plant or plant cell is the general appearance, health, visual symptoms of damage, such as wilting and oxidation of leaves, or the concentration of a metabolite. Methods and devices for selection are familiar to those skilled in the art and include GC (gas chromatography), LC (liquid chromatography), HPLC (high performance liquid chromatography (pressure)), MS (mass spectrometry), spectroscopy of NMR (nuclear magnetic resonance), IR (infrared) spectroscopy, photometric methods, etc., and combinations of these methods. Reproduction is also a common knowledge for those skilled in the art. It is understood as the directed and stable incorporation of a particular attribute or trait in a plant or plant cell. The various stages of reproduction are characterized by well-defined human intervention such as selecting crossing lines, directing pollination of parent lines, or selecting progeny plants
appropriate Different measures of reproduction can be taken, depending on the desired properties. All techniques are well known to a person skilled in the art and include, for example, but not limited to hybridization, inbreeding, hybridization reproduction, multiline reproduction, variety of mixtures, inter-specific hybridization, aneuploid techniques, etc. Hybridization techniques may also include sterilizing plants to produce male or female sterile plants by mechanical, chemical or biochemical means. The cross-pollination of a male sterile plant with pollen from a different line ensures that the genome of the sterile male is that the female fertile plant will uniformly obtain properties of both of the parental lines. The seeds and transgenic plants according to the invention can therefore be used for the reproduction of improved plant lines, which can increase the effectiveness of conventional methods such as treatment with herbicides or pesticides or which allow to distribute with such methods due to its modified genetic properties. Alternatively, new crops with improved stress tolerance, preferably drought and temperature, can be obtained, which, due to their optimized genetic "equipment", produce a harvested product of better quality than the products they were not able to tolerate.
comparable adverse development conditions. The invention provides that the environmental aggression can be by salinity, drought, temperature, metals, chemicals, pathogenic and oxidative aggressions, or combinations thereof, preferably drought and / or temperature. The object of the invention is a transgenic plant cell, wherein the SRP (stress-related protein) is preferably selected from yeast, preferably Saccharomyces cerevisiae, or E. coli or a plant. The object of the invention is a transgenic plant cell, wherein the SRP (stress-related protein) is preferably selected from a plant, preferably Brassica napus, Glycine max, Zea mays or Oryza sativa or yeast, preferably Saccharomyces cerevisiae , or E. coli, more preferably from Brassica napus, Glycine max, Zea mays or Oryza sativa. The object of the invention is also a transgenic plant cell, wherein the nucleic acid encoding SPR is at least about 50% homologous to one of the nucleic acid according to SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487. In the cell of the transgenic plant of the invention, expression of the nucleic acid results in increased tolerance to an assault environmental when it
compare with a corresponding non-transformed wild-type plant cell. In the present, the environmental aggression is selected from the group consisting of salinity, drought, temperature, metals, chemicals, pathogenic and oxidative aggressions, or combinations thereof, preferably by drought and / or temperature. The term "expression" refers to the transcription and / or translation of a codogenic genetic segment or gene. As a rule, the resulting product is an mRNA or a protein. However, the expression products may also include functional RNAs such as, for example, antisense, nucleic acids, tRNAs, sRNAs, RNAs, RNAi, siRNA, ribozymes, etc. The expression can be systemic, local or temporary, for example, limited to certain cell types, tissue, organs or periods of time. 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" can be related to polynucleotides, nucleic acids, nucleic acid molecules, peptides, polypeptides and proteins, depending on the context in which the term "sequence" is used. The terms "he or she
"genes", "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "the nucleic acid molecule (s)" as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyrubonucleotides The terms refer only to the primary structure of the molecule, thus, the terms "the gene (s)", "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "the "Nucleic acid molecules" as used herein include both double and single strand DNA and RNA.These also include known types of modifications, eg, mutilation, "covers" substitutions of one or more of the nucleotides of natural origin with a Analogously, the DNA or RNA sequence of the invention comprises a coding sequence encoding the polypeptide defined herein A "coding sequence" is a nucleotide sequence. otide, which is transcribed into the mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The limits of the coding sequence are determined by a translation start codon at the 5 'end and a translation stop codon at the 3' end. A coding sequence may include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, although the introns
They can also be presented under certain circumstances. For the purposes of the invention, as a rule, the plural is intended to encompass the singular and vice versa. In addition, the transgenic plant cell is derived from a monocotyledonous plant. Alternatively, the transgenic plant cell is derived from a dicotyledonous plant. Preferably, the transgenic plant cell is selected from the group consisting of corn, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, cottonseed, barley, cassava, pepper, sunflower, flax, borage, safflower, flaxseed, spring, rapeseed, wild rapeseed, marigold, solanaceous plants, potato, tobacco, eggplant, tomato, species Vicia, pea, alfalfa, coffee, cocoa, tea, Salix species, oil palm, coconut, herbs perennial, fodder and Arabidopsis thaliana. In addition, the transgenic plant cell of the present invention can be derived from a gymnosperm plant. Preferably, the plant is selected from the group of spruce, pine and spruce. The invention further provides a seed produced by a transgenic plant transformed by an SRP-encoding nucleic acid, wherein the plant is subjected to actual cross-breeding for increased tolerance to environmental aggression when compared to a wild-type plant cell. The transgenic plant could be a
monocotyledonous plant, a dicotyledonous or gymnosperm. The invention also provides a seed produced by a transgenic plant expressing an SRP wherein the plant is subjected to actual cross for increased tolerance to environmental aggression when compared to a wild-type plant cell. The invention pertains to a seed produced by a transgenic plant, wherein the seed is genetically homozygous for a transgene that confers an increased tolerance to environmental aggression when compared to a wild-type plant. The invention further provides an agricultural product produced by any of the transgenic plants described below, plant parts such as leaves, petals, anthers, roots, tubers, stems, buds, flowers or seeds. The invention further provides an isolated recombinant expression vector comprising a nucleic acid encoding SRP. The invention further provides a method for producing a transgenic plant with a SRP-encoding nucleic acid, wherein the expression of the nucleic acid in the plant results in tolerance and / or increased resistance to environmental aggression when compared to a plant cell of corresponding wild type, not transformed, comprising:
a) transforming a plant cell with an expression vector including an SRP coding nucleic acid selected from the group comprising the nucleic acid according to SEQ. FROM IDENT. DO NOT. : 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97 , 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197 , 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255 , 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305 , 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355 , 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405 , 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455 , 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505 , 507, 509, 511, 513, 515, 517 , 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567 , 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617 , 619, 621,
623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645,
647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669,
671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693,
695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717,
719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741,
743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765,
767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789,
791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813,
815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855,
859, 861, 863, 865, 867, 869, 871, 873, 875, 877,
881, 883, 885, 887, 889, 891, 893, 895, 897, 899,
903, 905, 907, 909, 911, 913, 915, 917, 919, 921,
925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965,
969, 971 and / or 973 / homologs or parts thereof and b) generate from the plant cell a transgenic plant with an increased tolerance to environmental aggression when compared to a corresponding non-transformed wild-type plant. With respect to the invention described herein, "transformed or transgene" means all those plants or parts thereof which have been produced by genetic manipulation methods and in which either
a) one or more genes, preferably encoded by one or more nucleic acid sequences as described in
SEC. FROM IDENT. DO NOT. : 1, 5, 9, 13, 17, 21, 25, 29, 33, 37,
41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93,
97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141,
145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185,
189, 193, 197, 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233 235, 237, 239,
241, 243, 245, 247, 249, 251, 253, 255 (257, 259, 261, 263,
265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287,
289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311,
313, 315, 317, 319, 321, 323, 325, 327 (329, 331, 333, 335,
337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359,
361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,
385, 387, 389, 391, 393, 395, 397, 399 (401, 403, 405, 407,
409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431,
433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455,
457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479,
481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503,
505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527,
529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551,
553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575,
577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599,
601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623,
625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647,
649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671,
673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695,
697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719,
721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767,
769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789,
791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811,
813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835,
837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881,
883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903,
905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925,
927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947,
949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973 and / or a homologue thereof, or b) a genetic regulatory element, eg, a promoter, the which is fionally linked, for example to the nucleic acid sequence as described in SEQ. FROM
IDENT. DO NOT. : 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97 101
105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145,
149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,
263, 265, 267, 269, 271, 273, 275 277, 279, 281, 283,
285, 287, 289, 291, 293, 295, 297 299, 301, 303, 305,
307, 309, 311, 313, 315, 317, 319 321, 323, 325, 327,
329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349,
351, 353, 355, 357, 359, 361, 363 365, 367, 369, 371,
373, 375, 377, 379, 381, 383, 385 387, 389, 391, 393,
395, 397, 399, 401, 403, 405, 407 409, 411, 413, 415,
417, 419, 421, 423, 425, 427, 429 431, 433, 435, 437,
439, 441, 443, 445, 447, 449, 451 453, 455, 457, 459,
461, 463, 465, 467, 469, 471, 473 475, 477, 479, 481,
483, 485, 487, 489, 491, 493, 495 497, 499, 501, 503,
505, 507, 509, 511, 513, 515, 517 519, 521, 523, 525,
527, 529, 531, 533, 535, 537, 539 541, 543, 545, 547,
549, 551, 553, 555, 557, 559, 561 563, 565, 567, 569,
571, 573, 575, 577, 579, 581, 583 585, 587, 589, 591,
593, 595, 597, 599, 601, 603, 605 607, 609, 611, 613,
615, 617, 619, 621, 623, 625, 627 629, 631, 633, 635,
637, 639, 641, 643, 645, 647, 649 651, 653, 655, 657,
659, 661, 663, 665, 667, 669, 671 673, 675, 677, 679,
681, 683, 685, 687, 689, 691, 693 695, 697, 699, 701,
703, 705, 707, 709, 711, 713, 715 717, 719, 721, 723,
725, 727, 729, 731, 733, 735, 737 739, 741, 743, 745,
747, 749, 751, 753, 755, 757, 759 761, 763, 765, 767,
769, 771, 773, 775, 777, 779, 781 783, 785, 787, 789,
791, 793, 795, 797, 799, 801, 803 805, 807, 809, 811,
813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833,
835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855,
857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877,
879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899,
901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921,
923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943,
945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965,
967, 969, 971 and / or 973 and / or a homologue thereof, or (a) and (b) does not occur or present in their natural genetic environment or has been or has been modified by genetic manipulation methods, it is 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 genetic, natural environment of the nucleic acid sequence is still preferably at least partially preserved. The environment flanks the nucleic acid sequence on at least one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably 1000 bp, very particularly preferably at least 5000 bp. In such a method to produce a transgenic plant comprising an SRP, the acid
nucleic acid encoding SRP is selected from the group comprising the nucleic acid according to SEQ. FROM IDENT. DO NOT. (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs of the sequences mentioned above. In addition, the SRP coding nucleic acid used in the method is at least about 50% homologous to one of the nucleic acid according to SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487. A plant or plant cell is considered a "real cross" for a particular trait if it is genetically homozygous for that trait to the extent that, when the actual cross-pollinated plant is self-pollinated, a significant amount of segregation independent of the progeny trait is not observed. In the present invention, the trait is raised from the transgenic expression of one or more DNA sequences introduced into a plant cell or plant. The present invention also provides methods for modifying the stress tolerance of a plant comprising, modifying the expression level of an SRP nucleic acid in the plant. The invention provides a method for producing a transgenic plant with a synthetic, novel or modified transcription factor that acts by increasing the transcription of an SRP gene. Theoretically, it is also possible to obtain a decrease in gene expression. A method to detect environmental aggression in
plant or plant cells comprising selecting plant cells for tolerance and / or increased resistance to environmental aggression when compared to conditions without stress is also within the scope of the invention. In addition, a method for selecting plant cells or plants for tolerance and / or increased resistance to environmental aggression that comprises selecting plant cells under stress conditions when compared to non-stress conditions is encompassed by the invention. The present invention also encompasses a method of reproducing plant or plant cells towards tolerance and / or increased resistance to environmental aggression which comprises selecting plant cells under stress conditions for tolerance and / or increased resistance to environmental aggression when compare with stress-free conditions and select those with tolerance and / or increased resistance to environmental aggression. The present invention also encompasses the use of tolerance and / or increased resistance to environmental aggression and / or a nucleic acid encoding SRP selected from the group comprising the nucleic acid according to SEQ. FROM IDENT. DO NOT. (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs of the sequences mentioned above or parts thereof as markers for selection of plants or plant cells
with increased tolerance to environmental aggression. The present invention further encompasses the use of tolerance and / or increased resistance to environmental aggression and / or an SRP coding nucleic acid selected from the group comprising the nucleic acid according to SEQ. FROM IDENT. DO NOT. (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs of the sequences mentioned above or parts thereof as markers for stress detection in plants or plant cells. The present invention also provides methods for modifying stress tolerance of a forage plant comprising using a SRP coding nucleic acid sequence to identify individual plants in populations that secrete tolerance to increased or decreased environmental aggression (DNA marker). In the method for modifying stress tolerance of a plant, the SRP coding nucleic acid can be selected from the group comprising the nucleic acid according to SEC. FROM IDENT. DO NOT. (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs of the aforementioned sequences. In addition, the SRP coding nucleic acid used herein may be at least about 50% homologous to one of the nucleic acid according to SEQ. FROM IDENT. DO NOT. (4n + l) for n = 0 to 54 and
(2n + 1) for n = 110 to 487. Also, an expression vector as described in the present invention could be used in the method. In a variant method of modifying stress tolerance, the plant is transformed with an inducible promoter that directs the expression of the SRP. For example, the promoter is tissue specific. In a variant method, the promoter used is regulated evolutionarily. In a further embodiment, the method for modifying stress tolerance comprises one or more of the following steps: a) stabilizing a protein that confers the increased expression of a protein encoded by the nucleic acid molecule of the invention or the polypeptide of the invention having the activity mentioned herein to increase the tolerance and / or resistance to environmental aggression when compared to a plant cell of the wild-type non-transformed, corresponding: b) stabilize an mRNA that confers the increased expression of a protein encoded by the nucleic acid molecule of the invention or its homologs or of an mRNA encoding the polypeptide of the present invention having the aforementioned activity of tolerance and / or increased resistance to environmental aggression when compared to a plant cell of type wild no
transformed, corresponding. c) increasing the specific activity of a problem that confers the increased expression of a protein encoded by the nucleic acid molecule of the invention or of the polypeptide of the present invention or by decreasing the inhibitory regulation of the polypeptide of the invention; d) generating or increasing the expression of an endogenous or artificial transcription factor that mediates the expression of a protein by conferring the increased expression of a protein encoded by the nucleic acid molecule of the invention or of the polypeptide of the invention having the activity mentioned in the present tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell. e) stimulating the activity of a protein that confers the increased expression of a protein encoded by the nucleic acid molecule of the present invention or a polypeptide of the present invention having the activity mentioned herein by increasing the tolerance and / or resistance to environmental aggression by adding one or more factors of exogenous induction to organisms or parts thereof; f) expressing a transgenic gene that encodes a
protein that confers the increased expression of a polypeptide encoded by the nucleic acid molecule of the present invention or a polypeptide of the present invention, having the aforementioned activity of tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell; and / or g) increasing the number of copies of a gene that confers 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 the activity mentioned in present tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell; h) increasing the expression of the endogenous gene encoding the polypeptide of the invention or its homologue by adding positive expression or removing negative expression elements, for example, homologous recombination can be used to introduce similar regulatory elements for plants of the 35S enhancer within the promoter or to remove repressive elements that form regulatory regions. In addition, genetic conversion methods can be used to interrupt repressor elements or to improve the activity of positive elements-positive elements that
they can be randomly introduced into plants by T-DNA mutagenesis or they can be identified in Oel Transposon and lines, in which the positive elements have to be integrated near a gene of the invention, the expression of which is consequently improved; and / or i) modulating the growth conditions of the plant in such a way, that the expression or activity of the gene encoding the protein of the invention or the protein itself is improved; j) selecting organisms with especially high activity of the proteins of the invention from natural resources or mutagenesis and reproducing them in the target organisms, for example, elite cultures. Preferably, the mRNA is the nucleic acid molecule of the present invention and / or the protein that confers the increased expression of a protein encoded by the nucleic acid molecule of the present invention of the polypeptide having the activity mentioned herein is the polypeptide of the present invention, for example, conferring increased tolerance to environmental aggression. In general, the amount of mRNA, the polynucleotide or nucleic acid molecule in a cell or a compartment of an organism correlates with the amount of encoded protein and thus the total activity of
the protein encoded in the volume. The 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 to activate or inhibit cofactors. In addition, inhibitors of product and conclusion of enzymes are well known, for example, Zinder et al. "Enzyminhibitoren / Enzyme inhibitors". The activity of aforementioned proteins and / or polypeptide encoded by the nucleic acid molecule of the present invention can be increased in several ways. For example, activity in an organism or in part of it, such as a cell, increases as the number of genetic products increases, for example, increasing the rate of expression, introducing a stronger promoter, or increasing the stability of the promoter. MRNA expressed, thus increasing the speed of translation, and / or increasing the stability of the gene product, thus reducing the impaired proteins. In addition, the activity or circulation of enzymes can be influenced in such a way that a reduction or increase in the reaction rate or a modification (reduction or increase) of the affinity of substrate results is achieved. A mutation in the catalytic center of a polypeptide of the invention, for example, as an enzyme can modulate the rate of circulation of the enzyme, for example, an inactivation of an essential amino acid can
lead to a reduced or completely inactivated activity of the enzyme, or deletion or mutation (or inhibition of the substrate, if the substrate level is also increased). The specific activity of an enzyme of the present invention can be increased so that the rate of circulation is increased or the binding of a cofactor is improved. Improving the stability of the coding mRNA or protein may also increase the activity of a gene product. Stimulation of the activity is also under the scope of the term "increased activity". In addition, the regulation of the aforementioned nucleic acid sequences can be modified so that gene expression is increased. This can be achieved advantageously by means of heterologous regulatory sequences or by modifying, for example, mutating, natural regulatory sequences which are presented. The advantageous methods can also be combined with each other. In general, an activity of a gene product in an organism or part thereof, in particular in a plant cell, a plant, or a plant tissue or a part thereof or in a microorganism can be increased by increasing the amount of the mRNA of specific coding or the corresponding protein in the organism or part thereof. "Amount of protein or mRNA" is understood as meaning the number of polypeptide molecules or
mRNA molecules in an organism, a tissue, a cell or a cell compartment. "Increase" in the amount of a protein means the quantitative increase of the molecular number of the protein in an organism, a tissue, a cell or a cellular compartment or part thereof - for example, by one of the methods described herein afterwards - compared to a wild type, control or reference. The increase in molecular number preferably involves at least 1%, preferably more than 10%, more preferably to 30% or more, especially preferably to 50%, 70%, or more, very especially preferably to 100%, more preferably at 500% or more. However, a de novo expression is also considered as subject of the present invention. A modification, that is, an increase or decrease, can be caused by endogenous or exogenous factors. For example, an increase in activity in an organism or a part thereof can be caused by adding a genetic product or a precursor or an activator or an agonist to the media or nutrition or they can be caused by introducing such subjects into an organism, transient or stable. In one embodiment, the increase or decrease in tolerance and / or resistance to environmental aggression when compared to a wild-type plant cell is not
corresponding transformation in the plant or a part thereof, for example, in a cell, a tissue, an organ, an organelle, etc., is achieved by increasing the endogenous level of the polypeptide of the invention. Accordingly, in one embodiment of the present invention, the present invention relates to a process wherein the number of genetic copies of a gene encoding the polynucleotide or nucleic acid molecule of the invention is increased. In addition, the endogenous level of the polypeptide of the invention can, for example, be increased by modifying the transcriptional or translational regulation of the polypeptide. In one embodiment, the tolerance and / or increased resistance to environmental aggression in the plant or part thereof may be altered by objective or random mutagenesis of the endogenous genes of the invention. For example, homologous recombination can be used to introduce positive regulatory elements such as for 35S enhancer plants into the promoter or to remove repressor elements that form regulatory regions. In addition, methods similar to the genetic conversion described by Kochevenko and Willmitzer (Plant Physiol 2003 May; 132 (1): 174-84) and citations herein may be used to interrupt repressor elements or to enhance the activity of positive regulatory elements. In addition, positive elements can be randomly introduced into genomes (plants) by T-DNA or
Transposon mutagenesis and lines can be selected, in which positive elements have been integrated near a gene of the invention, by expression which is consequently improved. The. Activation of plant genes by random integrations of enhancer elements has been described by Hayashi et al., 1992 (Science 258: 1350-1353) or eigel et al., 2000 (Plant Physiol. 122, 1003-1013) and others cited in the present. Inverse genetic strategies to identify insertions (which eventually carry the activation elements) close to genes of interest have been described for several cases, for example, 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, the material from all the plants of a plant population mutagenized by large T-DNA or Transposon is harvested and the genomic DNA is prepared. Then, the genomic DNA is grouped following the specific architectures as described for example in Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290). The genomic DNA groups are then selected by specific multiple PCR reactions that detect the
combination of the insertional mutagen (e.g., T-DNA or Transposon) and the gene of interest. Therefore, PCR reactions are activated in the DNA groups with specific combinations of T-DNA or transposon limit primers and genetic specific primers. General rules for primer design can again be taken from Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290). To revise lower levels of DNA groups leads to the identification of individual plants in which the gene of interest is activated by the insertional mutagen. The improvement of positive regulatory elements or the disruption or weakening of negative regulatory elements can also be achieved by common mutagenesis techniques: The production of chemically mutated populations or by radiation is a common technique and is known to the skilled artisan. The methods for plants are described by Koorneef et al. 1972 and citations herein and by Lighner and Caspar in "Methods in Molecular Biology" Vol. 82. These techniques usually induce point mutations that may be identical in any known gene using methods such as TILLING (Colbert et al., 2001). Accordingly, the level of expression may be increased if the endogenous genes encoding a polypeptide that confers increased expression of the
The polypeptide of the present invention, in particular genes comprising the nucleic acid molecule of the present invention, are modified by homologous recombination. Methods Tilling or genetic conversion. The regulatory sequences can be operably linked in the coding region of an endogenous protein and control their transcription and translation or the stability or deterioration of the coding mRNA, or the expressed protein. In order to modify and control expression, promoter, UTRs, splice sites, processing signals, polyadenylation sites, terminators, enhancers, repressors, post-transcriptional or post-translational modification sites can be changed, added or modified. For example, the activation of plant genes by random integrations of enhancer elements has been described by Hayashi et al., 1992 (Science 258: 1350-1353) or eigel et al., 2000 (Plant Physiol. 122, 1003-1013). ) and others cited here. For example, the expression level of the endogenous protein can be modulated by replacing the endogenous promoter with a stronger transgenic promoter or by replacing the endogenous 3'UTR with a 3'UTR, which provides more stability without modifying the coding region. In addition, transcriptional regulation can be modulated by the introduction of an artificial transcription factor as described in the examples. Promoters
Alternatives, terminators and UTRs are described later. Activation of an endogenous polypeptide having aforementioned activity, for example conferring an increased tolerance to environmental aggression can also be increased by introducing a synthetic transcription factor which binds the closure to the coding region of the gene encoding the invention and activates his transcript. A zinc finger protein can be constructed, which comprises a specific DNA binding domain and an activation domain such as, for example, the VP16 domain or herpes simplex virus. The specific binding domain can be linked to the regulatory region of the region encoding the protein. The expression of the chimeric transcription factor in a plant leads to a specific expression of the protein of the invention, see for example, in WO01 / 52620, Oriz, Proc. Nati Acad. Sci. USA, 2002, Vol. 99, 13290 or Guan Proc. Nati Acad. Sci. USA 2002, Vol. 88, 13296. In a further embodiment of the method according to the invention is used in which one of the aforementioned genes, or one of the aforementioned nucleic acids, is mutated in a manner that the activity of the encoded gene products is influenced less by cellular factors, or not at all, compared to non-cellular proteins.
mutated For example, the well-known regulatory mechanisms of enzymatic activity are the mechanisms of substrate inhibition or feedback regulation. Modes and techniques for the introduction of substitutions, deletions and additions of one or more bases, nucleotides or amino acids of a corresponding sequence are described hereinafter in the corresponding paragraphs and references listed herein, for example in Sambrook et al., Molecular Cloning , Cold Spring Habour, NY, 1989. The person skilled in the art will be able to identify regulatory domains and regulatory binding sites by comparing the sequence of the nucleic acid molecule of the present invention or the expression product thereof. with the state of the art by means of computer software which comprises algorithms for the identification of binding sites and regulatory domains or by systematically introducing into a nucleic acid molecule or a protein mutations and evaluating those mutations which will lead to a specific activity increased or an activity increased by vol umen, in particular per cell. Therefore, a nucleic acid molecule of the invention or a polypeptide of the invention derived from a related organism in a distant, evolutionary manner, for example, using a prokaryotic gene in a prokaryotic host, is expressly expressed in a plant.
as in these cases the regulatory mechanism of the host cell may not weaken the activity (cellular or specific) of the gene or its expression product. The mutation is introduced in such a way that the production of the amino acids is not adversely affected. Less influence on the regulation of a gene or its genetic product is understood as meaning a reduced regulation of the enzymatic activity that leads to a specific or increased cellular activity of the gene or its product. An increase in enzymatic activity is understood as meaning an enzymatic activity, which increases by at least 10%, advantageously at least 20, 30 or 40%, especially advantageously by at least 50, 60 or 70% compared to the starting body. The invention provides that the above methods can be carried out in such a way that stress tolerance is increased. It is also possible to obtain a decrease in stress tolerance. The invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions or specific methods, etc., as such, but they may vary and numerous modifications and variations will be apparent herein for those skilled in the art. The technique. It will also be understood that the terminology used
in the present it is for the purpose of describing specific modalities only and is not intended to be limiting. The present invention also relates to isolated Stress Related Proteins (SRPs) which are selected from the group consisting of the SEC proteins. FROM IDENT. NO .: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94 , 98,
102 106, 110, 114, 118, 122, 126, 130, 134, 138, 142 150 154, 158, 162, 166, 170, 174, 178, 182, 186, 190 198 202, 206, 210, 214 and / or 218 and / or 222, 224, 226, 228, 230, 232, 234, 236, 238 242 244, 246, 248, 250, 252, 254, 256, 258, 260, 262 266 268, 270, 272, 274 , 276, 278, 280, 282, 284, 286 290 292, 294, 296, 298, 300, 302, 304, 306, 308, 310 314 316, 318, 320, 322, 324, 326, 328, 330, 332 , 334 338 340, 342, 344, 346, 348, 350, 352, 354, 356, 358 362 364, 366, 368, 370, 372, 374, 376, 378, 380, 382 386 388, 390, 392, 394 , 396, 398, 400, 402, 404, 40-6 410 412, 414, 416, 418, 420, 422, 424, 426, 428, 430 434 436, 438, 440, 442, 444, 446, 448, 450 , 452, 454 458 460, 462, 464, 466, 468, 470, 472, 474, 476, 478 482 484, 486, 488, 490, 492, 494, 496, 498, 500, 502 506 508, 510, 512 , 514, 516, 518, 520, 522, 524, 526 530 532, 534, 536, 538, 540, 542, 544, 546, 548, 550 554 556, 558, 560, 562, 564, 566, 568, 570 , 572, 574
578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972 and / or 974 and / or homologous thereof. Preferably, the Stress Related Proteins (SRP) isolated from the present invention are selected from yeast or E. coli. In addition, the present invention relates to nucleic acids encoding the isolated Stress Related Protein (SRP) selected from the group comprising the SEC nucleic acid. DE.IDENT. NO .: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45,
49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105,
109 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,
241 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 291, 293 , 295, 297, 299, 301, 303, 305, 307, 309, 311, 313 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337 339, 341, 343, 345 , 347, 349, 351, 353, 355, 357, 359, 361 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385 387, 389, 391, 393, 395, 397 , 399, 401, 403, 405, 407, 409 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433 435, 437, 439, 441, 443, 445, 447, 449 , 451, 453, 455, 457 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481 483, 485, 487, 489, 491, 493, 495, 497, 499, 501 , 503, 505 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 64 7, 649 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697 699 , 701, 703, 705, 707, 709, 711, 713, 715, 717, 719,
721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771,773,775,777,779,781,783,785,787,789,791,793,795,797,799,801,803,805,807,809,811,813,815,817,819 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973; and / or homologs thereof. Here, preferably, a nucleic acid encoding the Stress Related Protein (SRP) isolated encodes an SRP which is selected from yeast or E. coli and / or Brassica napus, Glycine max, Zea mays or Oryza sativa. The present invention provides stress-related genetic sequences, selected from the group consisting of the SEC nucleic acid. FROM IDENT. NO .: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93 , 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193 , 197, 201, 205, 209, 213 and / or 217 of yeast, preferably of Saccharomyces cerevisiae or E. coli. and / or 221, 223, 225, 227, 229, 231, 233, 235, 237,
239, 241, 243, 245, 247 249, 251, 253, 255, 257, 259,
261, 263, 265, 267, 269 271, 273, 275, 277, 279, 281,
283, 285, 287, 289, 291 293, 295, 297, 299, 301, 303,
305, .307, 309, 311, 313 315, 317, 319, 321, 323, 325,
327, 329, 331, 333, 335 337, 339, 341, 343, 345, 347,
349, 351, 353, 355, 357 359, 361, 363, 365, 367, 369,
371, 373, 375, 377, 379 381, 383, 385, 387, 389, 391,
393, 395, 397, 399, 401 403, 405, 407, 409, 411, 413,
415, 417, 419, 421, 423 425, 427, 429, 431, 433, 435,
437, 439, 441, 443, 445 447, 449, 451, 453, 455, 457,
459, 461, 463, 465, 467 469, 471, 473, 475, 477, 479,
481, 483, 485, 487, 489 491, 493, 495, 497, 499, 501,
503, 505, 507, 509, 511 513, 515, 517, 519, 521, 523,
525, 527, 529, 531, 533 535, 537, 539, 541, 543, 545,
547, 549, 551, 553, 555 557, 559, 561, 563, 565, 567,
569, 571, 573, 575, 577 579, 581, 583, 585, 587, 589,
591, 593, 595, 597, 599 601, 603, 605, 607, 609, 611,
613, 615, 617, 619, 621 623, 625, 627, 629, 631, 633,
635, 637, 639, 641, 643 645, 647, 649, 651, 653, 655,
657, 659, 661, 663, 665 667, 669, 671, 673, 675, 677,
679, 681, 683, 685, 687 689, 691, 693, 695, 697, 699,
701, 703, 705, 707, 709 711, 713, 715, 717, 719, 721,
723, 725, 727, 729, 731 733, 735, 737, 739, 741, 743,
745, 747, 749, 751, 753 755, 757, 759, 761, 763, 765,
767, 769, 771, 773, 775 777, 779, 781, 783, 785, 787,
789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809,
811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831,
833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853,
855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875,
877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897,
899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919,
921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941,
943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963,
965, 967, 969, 971 and / or 973 and / or homologs thereof, preferably from Brassica napus, Glycine max, Zea mays or Oryza sativa. Homologs of the aforementioned sequences can be advantageously isolated from yeasts, fungi, viruses, algae, bacteria, such as Acetobacter (sub-gene: Acetobacter) acetyl; Acidithiobacillus ferrooxidans;
Acinetobacter sp .; Actinobacillus sp; Aeromonas salmonicida;
Agrobacterium tumefaciens; Aquifex aeolicus; Arcanobacterium pyogenes; Aster yellows phytoplasma; Bacillus sp .;
Bifidobacterium sp .; Borrelia burgdorferi; Brevibacterium linens; Brucella melitensis; Buchnera sp .; Butyrivibrio fibrisolvens; Campylobacter jejuni; Caulobacter crescentus;
Chlamydia sp .; Chlamydophila sp .; Chlorobium limicola;
Citrobacter rodentium; Clostridium sp .; Comamonas testosteroni;
Corynebacterium sp .; Coxiella burnetii; Deinococcus radiodurans; Dichelobacter nodosus; Edwardsiella ictaluri;
Enterobacter sp .; Erysipelothrix rhusiopathiae; Escherichia coli; Flavobacterium sp .; Francisella tularensis; Frankia sp.
Cpll; Fusobacterium nucleatum; Geobacillus stearothermophilus;
Gluconobacter oxydans; Haemophilus sp .; Helicobacter pylori; Klebsiella pneumoniae; Lactobacillus sp .; Lactococcus lactis;
Listeria sp .; Mannheimia haemolytica; Mesorhizobium loti;
Methylophaga thalassica; Microcystis aeruginosa; Microsciila sp. PRE1; Moraxella sp. TA144; Mycobacterium sp.; Mycoplasma sp .; Neisseria sp .; Nitrosomonas sp .; Nostoc sp. PCC 7120; Novosphingobium aromaticivorans; Oenococcus oeni; Pantoea citrea; Pasteurella multocida; Pediococcus pentosaceus;
Phormidium foveolarum; Phytoplasma sp.; Plectonema boryanum;
Prevotella ruminicola; Propionibacterium sp .; Proteus vulgaris;
Pseudomonas sp .; Ralstonia sp .; Rhizobium sp .; Rhodococcus equi; Rhodothermus marinus; Rickettsia sp .; Riemerella anatipestifer; Ruminococcus flavefaciens; Salmonella sp .;
Selenomonas ruminantium; Serratia entomophila; Shigella sp .;
Sinorhizobium meliloti; Staphylococcus sp .; Streptococcus sp .;
Streptomyces sp .; Synechococcus sp .; Synechocystis sp. PCC 6803; Maritime thermotoga; Treponema sp .; Ureaplasma urealyticum; Vibrio cholerae; Vibrio parahaemolyticus; Xylella fastidiosa; Yersinia sp .; Zymomonas mobilis, more preferably Salmonella sp. or Escherichia coli or plants, preferably of yeasts such as of the genus Saccharomyces, Pichia, Candida, Hansenula, Torulopsis or
Schizosaccharomyces or plants such as Arabidopsis thaliana, corn, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, borage, safflower, flaxseed, spring, rapeseed, canola and wild rapeseed, cassava, pepper, sunflower , marigold, solanaceous plant such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, thick plants such as coffee, cocoa, tea, Salix species, trees such as oil palm, coconut, perennial herbs, such as gramineae and pointer, and forage crops, such as alfalfa and clover and spruce, pine or spruce for example. More preferably, homologs of the aforementioned sequences can be isolated from Saccharomyces cerevisiae, E. coli or plants, preferably Brassica napus, Glycine max, Zea mays or Oryza sativa. The stress-related proteins of the present invention are produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector, e.g., in a binary vector, the expression vector is introduced into a host cell, e.g., wild-type Arabidopsis thaliana NASC N906 or any other plant cell as described in the examples seen below, and the stress-related protein is expressed in such a host cell. Examples for binary vectors are pBIN19, pBHOl, pBinAR, pGPTV, pCAMBIA, pBIB-
HYG, pBecks, pGreen or pPZP (Haj ukiewicz, P. et al., 1994, Plant Mol. Biol., 25: 989-994 and Hellens et al, Trends in Plant Science (2000) 5, 446-451). Advantageously, the nucleic acid sequences according to the invention or the genetic construct together with at least one reporter gene are cloned into an expression cassette, which is introduced into the organism through a vector or directly into the genome. This reporter gene should allow easy detection by means of a growth, fluorescence, chemical, bioluminescence or resistance test or by means of a photometric measurement. Examples of reporter genes which may be mentioned are genes resistant to antibiotics or herbicides, hydrolase genes, fluorescence protein genes, bioluminescence genes, sugar or nucleotide metabolic genes or biosynthesis genes such as the Ura3 gene, the Ilv2 gene, the luciferase gene, the ß-galactosidase gene, the gfp gene, the 2-deoxyglucose-6-phosphate phosphatase gene, the ß-glucuronidase gene, the ß-lactamase gene, the neomycin phosphotransferase gene, the hygromycin gene phosphotransferase or the BASTA gene (= resistance to glufosinate). These genes allow the measurement and quantification of assays for transcription activity and thus the expression of genes. In this way, genomic positions can be identified, which exhibit different productivity.
In a preferred embodiment a nucleic acid construct, eg, an expression cassette, comprises upstream, ie, at the 5 'end of the coding sequence, a promoter and downstream, ie, the 3' end, a polyadenylation signal and optionally other regulatory elements which are operably linked to the intermediate coding sequence with the SEC nucleic acid. FROM IDENT. DO NOT. : (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487. By the operable connection, it is meant the sequential arrangement of the promoter, coding sequence, terminator and optionally, other regulatory elements , in such a way that each of the regulatory elements can fulfill its function in the expression of the coding sequence in a convenient manner. Preferred sequences for operable connection are target sequences to ensure subcellular localization in plastids. However, target sequences to ensure subcellular localization in the mitochondria, in the endoplasmic reticulum (= ER), in the nucleus, in the oily corpuscle or other compartments can be employed as well as translation promoters such as the 5 'leader sequence in viruses of tobacco mosaic (Gallie et al., Nucí Acids, Res. 15 (1987), 8693-8711). A nucleic acid construct, for example, an expression cassette may, for example, contain a promoter
constitutive or a tissue-specific promoter (preferably the USP or napin promoter) the gene that is expressed and the ER retention signal. For the retention signal ER the amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) or the amino acid sequence KKX (lysine-lysine-X-arrest, where X means other known amino acids) is preferably used. For expression in a prokaryotic or eukaryotic host organism, for example, a microorganism such as a fungus or an expression cassette plant is advantageously inserted into a vector such as by way of example a plasmid, a phage or other DNA which allows the optimal expression of the genes in the host organism. Examples of suitable plasmids are: in E. coli series pLG338, pACYC184, pBR such as, for example, pBR322 series, pUC such as pUC18 or pCU19 series, M113mp, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, PUR290, pIN-III113-Bl, gtll or pBdCl; in Streptomyces pILIOl, pIJ364, pJJ / 02 or pIJ361; in Bacillus pUBUO, pC194 or pBD214; in Corynebacterium pSA77 or pAJ667; in fungi pALSl, pIL2 or pBB116; other advantageous fungal vectors are described by Romans, M.A. et al., [(1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488] and by van den Hondel, C.A. .J.J. et al. [(1991) "Heterologous gene expression in filamentous fungi" as well as in More Gene Manipulations in Fungi [J. . Bennet & L.L.
Lasure, eds. , pp. 396-428: Academic Press: San Diego] and in "Gene transfer systems and vector development for filamentous fungi" [van der Hondel, C.A.M.J.J. & Punt, P.J. (1991) in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., pp. 1-28, Cambridge University Press. Cambridge], Examples of advantageous yeast promoters are 2μ ?, pAG-1, Yep6, Yepl3 or pEMBLYe23. Examples of algae or plant promoters are pGLV23, pGHlac +, pBIN19, pAK2004, pVKH or pDH51 (see Schmidt, R. and illmitzer, L., 1988). The vectors identified above or derived from the vectors identified above are a small selection of the possible plasmids. Additional plasmids are well known to those skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pou els P.H. et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Suitable plant vectors are described inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Ch. 6/7, p. 71-119. Advantageous vectors are known as shuttle vectors or binary vectors which replicate in E. coli and Agrobacterium. By "vectors" is meant with the exception of plasmids other vectors known to those skilled in the art such as by way of example phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons,
IS elements, plasmids, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally, chromosomal replication is preferred. In a further embodiment of the vector, the expression cassette according to the invention can also be advantageously introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism by way of heterologous or homologous recombination. This linear DNA can be composed of a linearized plasmid or only of the expression cassette as a vector or the nucleic acid sequences according to the invention. In a further advantageous embodiment, the nucleic acid sequence according to the invention can also be introduced into an organism or by itself. If, in addition to the nucleic acid sequence according to the invention, additional genes are introduced into the organism, all together with the reporter gene in a single vector or each single gene with a reporter gene in a vector in each case can be introduced into the organism, so that different vectors can be introduced simultaneously or successively. The vector advantageously contains at least one copy of the nucleic acid sequences according to the invention and / or the expression cassette (= construction
genetic) according to the invention. The invention further provides an isolated recombinant expression vector comprising an SRP nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to environmental aggression when compared to a wild-type variety of the host cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a loop of double-stranded DNA within which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell at the introduction of the host cell, and therefore replicate together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In
In general, expression vectors of utility in recombinant DNA techniques are frequently in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably since the plasmid is the most commonly used form of vector. However, the invention is intended to include other such forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which perform equivalent functions. The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells that are used for expression, which is operably linked to the nucleic acid sequence that is expressed. As used herein, with respect to a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest binds to the regulatory sequence (s) in a manner which allows expression of the sequence of nucleotide (for example, in an in vitro transcription / translation system or in a host cell
when the vector is reintroduced into the host cell). The term "regulatory sequence" is intended to induce promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, eds. Glick and Thompson, chapter 7, 89-108, CRC Press: Boca Raton, Florida, including references herein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired polypeptide, etc. The expression vectors of the invention can be introduced into host cells whereby polypeptides or peptides are produced, including polypeptides or fusion peptides, encoded by acids as described herein (eg, SRPs, mutant forms of SRPs, polypeptides of fusion, etc.). The recombinant expression vectors of the invention can be designed for expression of SRPs in cells
prokaryotic or eukaryotic. For example, SRP genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romans, MA et al., 1992, Foreign gene expression in yeast: a review, Yeast 8: 423-488, van den Hondel, CAMJJ et al., 1991, Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, JW Bennett &LL Lasure, eds., p 396- 428: Academic Press: San Diego, and van den Hondel, CAMJJ &Punt, PJ, 1991, Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, JF et al., Eds., P. 1-28, Cambridge University Press: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251), ciliates of the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecio , Colpidium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euoplotes, Engelmanie lla, and Stylonychia, especially of the genus Stylonychia lemnae with vectors following a transformation method as described in PCT Application No. WO 98/01572, and multicellular plant cells (See Schmidt, R. and Illmit zer, L., 1988 , High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants, Plant Cell Rep. 583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton,
Florida, chapter 6/7, S. 71-119 (1993); F.F. hite, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung und R. Wu, 128-43, Academic Press: 1993; Protykus, 1991, Annu. Rev. Plant Physiol. Plant olec. Biol. 42: 205-225 and references cited therein) or mammalian cells. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press: San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Expression of polypeptide in prokaryotes is most frequently carried out with vectors containing constitutive or inducible promoters that direct the expression of any fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded herein, usually to the amino terminus of the recombinant polypeptide, but also to the C terminus or combined within suitable regions in the polypeptides. Such fusion vectors normally serve three purposes: 1) to increase the expression of a recombinant polypeptide; 2) increase the solubility of a recombinant polypeptide; and 3) add in the purification of a recombinant polypeptide by acting as a ligand in
affinity purification. Frequently, in the fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion portion and the recombinant polypeptide allowing the separation of the recombinant polypeptide from the fusion portion subsequent to purification of the polypeptide from fusion. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. By way of example, the expression cassette can be installed in the transformation vector pRT ((a) Toepfer et al., 1993, ethods Enzymol., 217: 66-78; (b) Toepfer et al., 1987, Nucí. Res. 15: 5890 ff.). Alternatively, a recombinant vector (= expression vector) can also be transcribed and translated in vitro, for example, using the T7 promoter and the T7 RNA polymerase. Expression vectors employed in prokaryotes often make use of inducible systems with and without fusion proteins or fusion oligopeptides, wherein these fusions can occur in both N-terminal and C-terminal ways or in other useful domains of a protein. Such fusion vectors usually have the following purposes: i) increase the rate of RNA expression; ii.) increase the rate of protein synthesis achievable; iii.) increase the solubility of the protein;
iv.) or simplify the purification by means of a linker sequence usable by affinity chromatography. The proteolytic cleavage sites are also introduced proteolytically through fusion proteins, which allow unfolding of a portion of the protein and fusion purification. Such recognition sequences for proteases are recognized, for example, factor Xa, thrombin and enterokinase. Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which contains glutathione S-transferase (GST), maltose binding protein or protein A. In one embodiment, the SRP coding sequence is cloned into a pGEX expression vector to create a vector encoding a fusion polypeptide comprising, from the N-terminus to the C-terminus, X-site polypeptide of GST-thrombin cleavage. . The fusion polypeptide can be purified by affinity chromatography using glutathione-agarose resin. Recombinant PKSRP not combined with GST can be recovered by cleavage of the fusion polypeptide with thrombin. Other examples of E. coli expression vectors are pTrc [Amann et al., (1988) Gene 69: 301-315] and pET vectors
[Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Stratagene, Amsterdam, The Netherlands]. The target gene expression of the pTrc vector is based on transcription of host RNA polymerase from a hybrid trp-lac fusion promoter. Target gene expression from the pET lid vector is based on transcription from a T7 gnlO-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by BL21 (DE3) or HSM174 (DE3) host strains from a pro-phage? resident harboring a T7 gnl gene under the transcriptional control of the lacUV promoter 5. One strategy to maximize the expression of recombinant polypeptide is to express the polypeptide in a host bacterium with an impaired ability to proteolytically unfold the recombinant polypeptide (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence that is inserted into an expression vector so that the individual codons for each amino acid are preferably used in the bacterium chosen for expression, such as C. glutamicum (Wada et al., 1992, Nucleic Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences
of the invention can be carried out by standard DNA synthesis techniques. Other advantageous vectors for use in yeast are pYepSecl (Baldari, et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herso itz, (1982) Cell 30: 933-943), San Diego Corporation , CA): Vectors for use in filamentous fungus are described in: van den Hondel, CAMJJ & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi", in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., Eds. , pp. 1-28, Cambridge University Press: Cambridge. Alternatively, expression vectors of insect cells can also be advantageously used, for example, for expression in Sf9 cells. There are, for example, the vectors of the pAc series (Smith et al. (1983) Mol Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39). In addition, plant cells or algal cells can be advantageously used for gene expression. Examples of plant expression vectors can be found in Becker, D., et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197 or in Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucí. Acid Res. 12: 8711-8721.
In addition, nucleic acid sequences can also be expressed in mammalian cells, advantageously in non-human mammalian cells. Examples of corresponding expression vectors are pCD 8 and pMT2PC indicated in: Seed, B. (1987) nature 329: 840 or Kaufman et al. (1987) EMBO J. 6: 187-195). At the same time preferred promoters for use are of viral origin, such as by way of example, promoters of polyoma, adenovirus 2, cytomegalovirus or simian virus. Other prokaryotic and eukaryotic expression systems are indicated in chapters 16 and 17 of Sambrook et al., Molecular Cloning: A Laboratory Manual. 2a. ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. In a preferred embodiment of the present invention, SRPs are expressed in plants and plant cells such as cells from unicellular plants (e.g. , algae) (See Falciatore et al, 1999, Marine Biotechnology 1 (3): 239-251 and references herein) and plant cells of higher plants (e.g., spermatophytes, such as forage plants). An SRP can be "introduced into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection and the like." A transformation method known to those skilled in the art is the immersion of a plant.
flourishing in a solution of Agrobacteria, where the Agrobacteria contains the SRP nucleic acid, followed by reproduction of the transformed gametes. Other suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2a, ed., Cold Spring Habor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and other laboratory manuals such as Methods in Molecular Biology, 1995, Vol. 44. Agrobacterium protocols, ed: Gartland and Davey , Humana Press, Totowa, New Jersey. Since tolerance to abiotic aggression is a general trait desired to be inherited in a wide variety of plants such as corn, wheat, rye, oats, triticale, rice, barley, soybeans, peanuts, cotton, rapeseed and canola, cassava , pepper, sunflower and marigold, solanaceous plants such as potatoes, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, thick plants (coffee, cocoa, tea), species Salix, trees (oil palm, coconut), perennial herbs and forage crops, these forage plants are also preferred target plants for genetic engineering as a further embodiment of the present invention. Forage crops include, but are not limited to Wheatgrass, Canarygrass, Bromegrass, ildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin,
Cuernecillo, Hybrid Clover, Red Clover and Meliloto. In one embodiment of the present invention, transfection of an SRP in a plant is achieved by Agrobacterium-mediated gene transfer. The transformation of the Agrobacterium-mediated plant can be carried out using, for example, GV3101 (pMP90) (Koncz and Schell, 1986, Mol.Gen.Genet., 204: 383-396) or strain LBA4404 Clontech) Agrobacterium tumefaciens. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucí Acids Res 13: 4777-4788; Gelvin Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd ed. : Kluwer Academic Publ., 1995. - in Sect., Ringbuc Zentrale Signatur: BTll-P ISBN 0-7923-2731-4; Glick, Bernard R.; Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Mouth Mouse: CRC Press, 1993 360 S. ISBN 0-8493-5164-2). For example, rape seed can be transformed by cotyledonary or hypocotyledon transformation (Molones et al., 1989, Plant Cell Report 8: 238-242; De Block et al., 1989, Plant Physiol. 91: 694-701). The use of antibiotics for Agrobacterium and plant selection depends on the binary vector and is usually done using kanamycin as a selectable plant marker. The genetic transfer mediated by agrobacterium to flax can be done using, for example, a technique
described in European Patent No. 0424 047, US Patent No. 5,322,783, European Patent No. 0397 687, US Patent No. 5,376,543 or US Patent No. 5,169,770. Corn transformation can be achieved by particle bombardment, DNA incorporation mediated by polyethylene glycol or by silicon carbide fiber technique. (See for example, Freeling and albot "The maize handbook" Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of transformation is found in U.S. Patent No. 5,990,387 and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256. In accordance with the present invention, the introduced SRP can be stably maintained in the plant cell if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes. Alternatively, the introduced SRP may be presented in a vector without extra-chromosomal replication and is temporarily expressed or temporarily activated. In one embodiment, a homologous recombinant microorganism can be created wherein the SRP is integrated into a chromosome, a vector is prepared which contains at least a portion of an SRP gene into which a deletion, addition or substitution has been introduced for thereby altering, for example, functionally affecting, the SRP gene. From
Preferably, the SRP gene is a yeast, SRP E. coli gene, but can be a homologue from a related plant or even from a mammalian or insect source. In one embodiment, the vector is designed such that, in homologous recombination, the endogenous SRP gene is functionally affected (i.e., it no longer encodes a functional polypeptide, also indicated as an inactivation vector). Alternatively, the vector can be designed such that, in homologous recombination, the endogenous SRP gene is mutated or otherwise altered, but it encodes even a functional polypeptide (eg, the upstream regulatory region can be altered to alter mode expression of endogenous SRP). To create a point mutation by homologous recombination, the DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al., 1999, Nucleic Acids Research 27 (5): 1323-1330 and Kmiec, 1999 Gene therapy American Scientist 87 (3): 240-247). Homologous recombination procedures in Physcomitrella patents are also known in the art and are contemplated for use herein. Although in the homologous recombination vector, the altered portion of the SRP gene is flanked at the 5 'and 3' ends by an additional nucleic acid molecule of the SRP gene to allow homologous recombination that occurs
between the exogenous SRP gene transported by the vector and an endogenous SRP gene, in a microorganism or plant. The additional flanking SRP nucleic acid molecule is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several hundred base pairs up to kilobases of flanking DNA (both of the 5 'and 3' ends) are included in the vector. See for example, Thomas, KR, and Capecchi, MR, 1987, Cell 51: 503 for a description of the homologous recombination vectors or Strepp et al., 1998, PNAS, 95 (8): 4368-373 for recombination based on CDNA in Physcomitrella patents). The vector is introduced into a microorganism or plant cell (for example, through DNA mediated by polyethylene glycol) and cells in which the introduced SRP gene has recombined in an homologous manner with the endogenous SRP gene are selected using known techniques in the art. In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow regulated expression of the introduced gene. For example, inclusion of an SRP gene in a vector places it under the control of the lac operon that allows the expression of the SRP gene only in the presence of IPTG. Such regulatory systems are well known in the art. If it occurs in a vector without extra-chromosomal replication or a vector that is integrated into a chromosome,
the SRP polynucleotide preferably resides in a plant expression cassette. A plant expression cassette preferably contains regulatory sequences capable of driving expression in cells of plants that are operably linked so that each sequence can fulfill its function, for example, transcription termination by polyadenylation signals. Preferred polyadenylation signals are those that originate from t-DNA of Agrobacterium tumefaciens such as gene 3 known as octopine synthase from the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J.3: 835) or functional equivalents of the same, but also other terminators functionally active in plants are suitable. Since the gene expression of the plant is not very frequently limited in transcriptional levels, a plant expression cassette preferably contains other sequences operably linked as translation enhancers such as the extraordinary sequence containing the 5 'untranslated leader sequence from of tobacco mosaic virus by improving the polypeptide by RNA ratio (Gallie et al., 1987, Nucí Acids Research 15: 8693-8711). Examples of plant expression vectors include those detailed in: Becker, D. et al., 1992, New plant binary vectors with selectable markers located proximal to the left border, Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W., 1984, Binary
Agrobacterium vectors for plant transformation, Nucl. Acid Res. 12: 8711-8721; and Vectors for Gene Transfer in Higher Plants; in: Transgenic Plant, Vol. 1, Engineering and Utilization, eds. : Kung and R. Wu, Acadertiic Press, 1993, S. 15-38. "Transformation" is defined herein as a process for introducing heterologous DNA into a plant cell, plant tissue or plant. This can occur under natural or artificial conditions using various methods well known in the art. The transformation can be based on any known method for the insertion of nucleic acid sequences into an approbable or eukaryotic host cell. The method is selected based on the host cell that is transformed and may include, but is not limited to, viral infection, electroporation, lipofection and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicated plasmid or as part of the host chromosome. These also include cells which temporarily express the inserted DNA or RNA for limited periods of time. The cells of transformed plants, plant tissue, or plants are understood to encompass not only the final product of a transformation process, but also the transgenic progeny of the
same. The terms "transformed", "transgenic" and "recombinant" refer to a host organism such as a bacterium or a plant within which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be presented as an extrachromosomal molecule. Such an extrachromosomal molecule can self-replicate. Transformed cells, tissues or plants are understood to encompass not only the final product of a transformation process, but also transgenic progeny thereof. An "untransformed", "non-transgenic" or "non-recombinant" host refers to a wild type organism, for example, a bacterium or plant, which does not contain the heterologous nucleic acid molecule. A "transgenic plant" as used herein, refers to a plant which contains a foreign nucleotide sequence, inserted into any of its nuclear genome or organellar genome. The descendant generations are also included, that is, the IT, T2 and consecutively generations or BC1-, BC2- and consecutively generation as well as hybrids thereof with non-transgenic or other transgenic plants. The host organism (= transgenic organism)
advantageously contains at least one copy of the nucleic acid according to the invention and / or the nucleic acid construct according to the invention. In principle, all plants can be used as a host organism. Preferred transgenic plants are selected for example from the families Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae, Papveraceae, Rosaceae, Salicaceae, Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae, Iridaceae, Liliaceae, Orchidaceae, Gentianaceae, Labiaceae, agnoliaceae,
Ranunculaceae, Carifolaceae, Rubiciaceae, Scrophulariaceae, Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae or Poaceae and preferably a plant selected from the group of the families Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae, Papveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Preferred plants are forage plants such as plants advantageously selected from the peanut, oilseed rape, cañola, sunflower, safflower, olive, sesame, hazelnut, almond, avocado, berry, pumpkin / chayote, flaxseed, soybean, pistachio, borage, corn, wheat, rye, oats, sorghum and millet, triticale, rice, barley, cassava, potato, sugar cane, eggplant, alfalfa and perennial herbs and forage plants, oil palm, vegetables (cabbage, root vegetables,
tubers, pod vegetables), vegetables that bear fruits, onions, vegetables with leaves and stems vegetables), buckwheat, ground artichoke, garbanzo beans, carob, lentil, dwarf beans, lupine, clover and alfalfa to mention just a few they. In a preferred embodiment, the host plant is selected from the families Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae, alvaceae, 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 of a plant selected from the group of the families Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae, Papveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Forage crops are preferred in particular plants mentioned hereinabove as host plants such as the families and genera mentioned above for example, the species Anacardium occidentale, Calendula officinalis, Carthamus tinctorius, Cichorium intybus, Cynara scolymus, Helianthus agnus are preferred. , Tagetes lucida, Tagetes erecta, Tagetes tenuifolia; Daucus carota; Corylus avellana, Corylus column,
Borago officinalis; Brassica napus, Brassica rapa ssp., Sinapis arvensis Brassica júncea, Brassica júncea var. Júncea, Brassica júncea var. crispifolia, Brassica júncea var. Foliosa, Brassica nigra, Brassica sinapioides, Elanosinapis 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, maritime Beta, Beta vulgaris var. perennis, Beta vulgaris var. conditive, Beta vulgaris var. esculenta, Maximum Cucurbit, Mixed Cucurbit, Cucurbita pepo, Cucurbita moschata, Otea 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 soybean, Glycine gracilis, Glycine hispida, Phaseolus max, Soybean hispida, Soy max, Cocos nucifera, Pelargonium grossularioides, Oleum coceas, Laurus nobilis, Persea americana, Arachis hypogaea, Linum usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum perennial, Linum perenne var.
lewisii, Linum pratense, Linum trigynum, Punic granatum, Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum, Gossypium thurberi, Musa nana, Musa acuminata, Musa paradisiaca, Musa spp., Elaeis guineensis, Papaver oriéntale, Papaver rhoeas, Papaver dubium , Sesamum 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 sativa, 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 cemuum, Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guiñéense, 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 hybemum, 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. The anacardiaceae such as the genus Pistacia, angifera, Anacardium, for example, the species Pistacia vera [pistaches, Pistazie], Mangifer indica [Mango] or Anacardium occidentale [Cashew]; Asteraceae such as the genus Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana, for example, the species Calendula officinalis [Calendula]. Carthamus tinctorius [safflower], Centaurea cyanus [cornflower], Cichorium intybus [blue daisy], Cynara scolymus [Artichoke], Helianthus agnus [sunflower], Lactuca sativa, Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp. Sativa Subs. Romana, Locusta communis, Valeriana locusta [lettuce], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [Calendula]; Apiaceae such as the Daucus genera, for example, the species Daucus carota [carrot]; Betulaceae such as the genus Corylus for example, the species Corylus avellana or Corylus column [hazelnut]; Boraginaceae such as the genus Borraja, for example, the species Borago officinalis [borraja]; Brassicaceae such as the genus
Brassica, Melanosinapis, Sinapis, Arabadopsis, for example, the species Brassica napus, Brassica rapa ssp. [canola, oilseed rape, rapeseed], Sinapis arvensis Brassica júncea, Brassica júncea var. júncea, Brassica júncea var. crispifolia, Brassica júncea var. foliosa, Brassica nigra, Brassica sinapioides, Melanosinapis comraunis [mustard], Brassica olerácea [fodder beet] or Arabidopsis thaliana; Bromeliaceae such as the genus Anana, Bromelia for example, the species Anana comosus, Ananas ananas or Bromelia comosa [pineapple]; Caricaceae such as the genus Carica for example, the species Carica papaya [papaya]; Cannabaceaetal as the genus Cannabis for example, the species Cannabis sative [hemp], Convolvulaceae such as the genus Ipomea, Convolvulus, for example, the species Ipomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus [sweet potato, Man of the Heat, wild potato], Chenopodiaceae such as the genus Beta, that is, the species Beta vulgaris, Beta vulgaris var. Very high, Beta vulgaris var. Vulgaris, maritime Beta, Beta vulgaris var. Perennis, Beta vulgaris var. Conditive or Beta vulgaris var. esculenta [sugar beet]; Cucurbitaceae such as the genus Cucurbita for example, the species Cucurbita maximus, Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [squash, chayote]; Elaeagnaceae such as the genus
Elaeagnus, for example, the species Olea europaea [oliva]; Ericaceae such as the genus Kalmia for example, the species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Western Kalmia, Cistus chamaerhodendros or Kalmia lucida [American laurel, broadleaf laurel, mountain laurel, wood for spoons, laurel swamp, alpine laurel, marsh laurel, western marsh laurel, mud laurel]; Euphorbiaceae such as the genus Manihot, Janipha, Jatropha, Ricinus for example, the most useful Manihot species, Janipha manihot., Jatropha manihot., Manihot aipil, Manihot sweets, manihot manihot, Manihot melanobasis, Manihot esculenta [yucca, arruruz, tapioca, yucca ] or Ricinus communis [castor bean, Castor OilBush, Castor Oil Plant, Palma Christi, Gonder Tree]; Fabaceae such as the genus Pisum, Albizia, Cathormion, Feuillea, Inga, Pithecolobium, acacia, Mimosa, Medicajo, Glycine, Dolichos, Phaseoulus, Soja, for example, the species Pisum sativum, Pisum arvense, Pisum humile [chícharo], Albizia Albizia julibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis, Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuillea berteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobium fragrans, 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 [Campeche hybrid wood, Silk tree, East Indian walnut], Medicago sativa, Medicago falcata, Medicago varia [alfalfa], Glycine max Dolichos soybean, Glycine gracilis, Glycine hispida, Phaseolus max, Soybean hispida or Soy max [soybean]; Geraniacea such as the genus Pelargonium, Cocos, Oleum for example, the species Cocos nucifera, Pelargonium grossularioides or Oleum cocois [coco]; Gramineae such as the genus Saccharum for example the species Saccharum officinarum; Juglandaceae such as the genus Juglans, Wallia for example, the species Junglans regia, Juglands ailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra [walnut, black walnut, common walnut, Caucasian walnut, for example, the laurel species Laurus nobilis [berry, bay, bay berry, sweet berry], Persea americana Persea americana, Persea gratissima or Persea persea [avocado]; Leguminosae such as the genus Arachis for example, the species Arachis hypogaea [peanut]; Linaceae such as the genus Linum, Adenolinum for example 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 perennial, Linum perennial var. lewisii, Linum pratense or Linum trigynum [flax, flaxseed]; Lythrarieae such as the Punic genus, for example the species Punic granatum [pomegranate]; Malvacea such as, for example, the species Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum or Gossypium thurberi [cotton]; Musaceae such as the genus Musa for example, the species Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [banana]; Onagraceae such as the genus Camissonia, Oenothera for example, the species Oenothera biennis or Camissonia brevipes [spring, evening spring]; Palmaea such as the genus Elacis for example, the species Elaeis guineensis [oil palm]; Papaveraceae such as the genus Papaver for example, the species Papaver oriéntale, Papaver rhoeas, Papaver dubium [poppy, oriental poppy, common poppy, field poppy, Shirley poppies, field poppy, long-headed poppy, long-poppy poppy]; Pedaliaceae such as the genus Sesamum for example, the species Sesamum indicum [sesame]; Piperaceae such as the genus Piper, Artanthe, Peperomia, Steffensia for example, the species Piper aduncum, Piper amalago, Piper angustifolium, Piper auritum, Pipier betel, piper cubeba, Piper longum, Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe elongata, Peperomia elongata, Piper elongatum, Steffensia elongata. [Pepper of
Cayenne, wild pepper] Poaceae such as the genus Hordeum, Sécale, Oats, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea, Triticum, for example, the species Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea, Triticum , for example, 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, pod barley , meadow barley], Sécale cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. Sativa, Avena hybrida [oats], 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 guiñéense, Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcus halepensis, Sorghum miliaceum millet, Panicum militaceum [sorghum, mojo], 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, common bread wheat, common wheat], Proteaceae such as the genus Macadamia, for example, the species Macadamia intergrifolía
[macadamia]; Rubiaceae such as the genus Coffea for example, the species Cofea spp., Coffea arabica, Coffea canephora or Coffea liberica [coffee]; Scrophulariaceae such as the genus Verbascum for example, 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, mullein of pine, nettle mullein, mullein with dense flowers, silver mullein, longleaf mullein, white mullein, dark mullein; Greek mullein, orange mullein, purple mullein, grayish mullein, common mullein]; Solanaceae such as the genus Capsicum, Nicotiana, Solanum, Lycopersicon, for example, the species Capsicum Nahum, Capsicum annuum var. glabriusculum, Capsiucum frutescens [pepper], Capsicum annuum [piminger], Nicotiana tabacum, Nicotiana alata, Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotiana obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana rustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [ papa], Solanum melongena [aubergine] (Lycopersicon esculentum, Lycopersicon lycopersicum., Lycopersicon periforme, Solanum integrifolium or Solanum lycopersicum [tomato]; Sterculiaceae such as the genus Theobroma, for example, the species Theobroma cacao [cocoa]; Theaceae as the genus
Camellia, for example, the species Camellia sinensis) [tea]. The introduction of the nucleic acids according to the invention, the expression cassette or the vector within organisms, plants for example, can in principle be done by all methods known to those skilled in the art. The introduction of the nucleic acid sequences gives rise to recombinant or transgenic organisms. In the case of microorganisms, those skilled in the art can find appropriate methods in the textbooks by Sambrook, J. et al. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by F.M. Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by D.M. Glover et al., DNA Cloning Vol. 1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press or Guthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press. The transfer of foreign genes into the genome of a plant is called transformation. In doing so, the methods described for the transformation and regeneration of plants from plant tissues or plant cells are used for transient or stable transformation. Suitable methods are transformation of protoplasts by
DNA incorporation induced by poly (ethylene glycol), the "biolistic" method using the cannon gene - indicated as the particle bombardment method, electroporation, incubation of dried embryos in DNA solution, microinjection and Agrobacterium-mediated gene transfer. The 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. u, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). Nucleic acids or the construct that is expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example, pBinl9 (Bevan et al., Nucí Acids Res. 12 (1984) 8711). The agrobacteria transformed by such vector can then be used in a known manner for the transformation of plants, in particular of forage plants such as for example tobacco plants, for example, soaking bruised leaves or leaves cut into pieces in an agrobacterial solution. and then cultivating them in appropriate media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hófgen and Willmitzer in Nucí. Acid Res. (1988) 16, 9877 or known inter alia from F.F. hite, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R, Wu,
Academic Press, 193, pp. 15-38. The agrobacteria transformed by an expression vector according to the invention can likewise be used in a known manner for the transformation of plants such as test plants such as Arabidopsis or forage plants such as cereal crops, corn, oats, rye, barley, wheat, soybeans, rice, cotton, sugar beet, sugar cane, sunflower, flax, hemp, potatoes, tobacco, tomatoes, carrots, peppers, oilseed rape, tapioca, cassava, arrowroot, marigold, alfalfa, lettuce and various trees, nuts and vine species, in particular forage plants containing oil, such as soybean, peanut, castor oil plant, sunflower, corn, cotton, flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius), or cocoa beans, for example, soaking bruised leaves or leaves cut into pieces in an agrobacterial solution and then growing them in suitable media. The cells of genetically modified plants can be regenerated by all methods known to those skilled in the art. Appropriate methods can be found in the publications indicated above by S.D. Kung and R. Wu, potrykus or Hófgen and Wilmit zer. Therefore, an additional aspect of the
invention relates to transgenic organisms transformed by at least one nucleic acid sequence, expression cassette or vector according to the invention as well as cells, cell cultures, tissue, parts - such as, for example, leaves, roots, etc., in the case of plant organisms - or reproductive material derived from such organisms. The terms "host organism", "host cell", "recombinant organism (host)", and "transgenic cell (host)" are used here interchangeably. Of course, these terms relate not only to the particular host organism or the particular target cell but also to the potential descendants or descendants of these organisms or cells. Since, due to mutation or environmental effects certain modifications may arise in successive generations, these descendants are not necessarily identical with the paternal cell, but nevertheless they are still encompassed by the term as it is used in the present. For purposes of the invention, "transgenic" or "recombinant" means with respect to for example a nucleic acid sequence, an expression cassette (= genetic construct, nucleic acid construct) or a vector containing the nucleic acid sequence according to the invention or an organism transformed by the nucleic acid, expression cassette or vector of
according to the invention all constructions produced by genetic engineering methods in which either a) the nucleic acid sequence described SEC. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 or its derivatives or parts thereof or b) a genetic control sequence functionally linked to the nucleic acid sequence described under (a), for example, a 3 'and / or 5' genetic control sequence such as a promoter or a terminator, or c) (a) and (b) are not found in their natural genetic environment or have been modified by genetic engineering methods, wherein the modification may by way of example be a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment means the natural genomic or chromosomal locus in the organism of origin or within the host organism or presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained at least in part. The environment borders the nucleic acid sequence on at least one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1,000 bp, more particularly preferably at least 5,000 bp. A cassette
of expression of natural origin - for example, the natural origin combination of the natural promoter of the nucleic acid sequence according to the invention with the genes? -8-desaturase,? -9-elongase and / or? -5-desaturase - transforms into a cassette of transgenic expression when the latter is modified by non-natural, synthetic ("artificial") methods such as by way of example a mutagenation. Appropriate methods are described by way of example in US 5,565,350 or WO 00/15815. Suitable organisms or host organisms for the nucleic acid, expression cassette or vector according to the invention are advantageously in principle all organisms, which are suitable for the expression of recombinant genes, as described above. Additional examples which may be mentioned are plants such as Arabidopsis, Asteraceae such as Calendula or forage plants such as soybean, peanut, castor oil plant, sunflower, flax, corn, cotton, flax, oilseed rape, coconut, oil palm , safflower (Carthamus tinctorius), or cocoa beans. A further object of the invention relates to the use of a nucleic acid construct, for example, an expression cassette, which contains DNA sequences encoding polypeptides of SEQ. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 or DNA sequences that
hybridize with it for the transformation of plant cells, tissues or parts of plants. In this way, depending on the choice of the promoter, the sequences of SEC. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487 can be expressed specifically in leaves, seeds, nodules, roots, stems or other parts of plant. Those transgenic plants that overproduce SEC sequences. FROM IDENT. DO NOT. : (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487, the reproductive material of the same, together with the cells of the plants, tissues or parts of it are an additional object of the present invention. The expression cassette or nucleic acid sequences or construct according to the invention, which contains sequences according to SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 can also be used for the transformation of the organisms identified by way of previous examples such as bacteria, yeast, fungus filamentous and plants. Within the framework of the present invention, tolerance and / or increased resistance to environmental aggression means, for example, the artificially acquired trait of increased biosynthetic performance due to functional overexpression of SEC sequences. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 coded by the SEC. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and
(2n + l) by n = 110 to 487 and / or homologs in the organisms according to the invention, advantageously in the transgenic plants according to the invention, by comparison with the initial non-genetically modified plants at least for the duration of at least one generation of plants. A constitutive expression of the exogenous sequences of the SEC. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 coded for the SEC. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 and / or 217 and / or homologs, is also advantageous. On the other hand, however, an inducible expression may seem desirable. The efficiency of the expression of the SEC sequences. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 coded by the SEC. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 and / or homologs can be determined, for example, in vitro spread of shoot meristem. In addition, an expression of the sequences of the SEC. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 coded by the SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs modified by nature and level and their effect on metabolic trajectory performance can be tested in test plants in greenhouse features. A further object of the invention comprises transgenic organisms such as transgenic plants
transformed by an expression cassette containing sequences of the SEC. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 according to the invention or DNA sequences that hybridize with it, as well as transgenic cells, tissue, parts and reproduction material of such plants. Particular preference is given in this case to transgenic forage plants such as, for example, barley, wheat, rye, oats, corn, soy, rice, cotton, sugar beet, oilseed rape and canola, sunflower, flax, hemp, thistle, potatoes, tobacco, tomatoes, tapioca, cassava, arrowroot, alfalfa, lettuce and several trees, nuts and species of vids. For the purposes of the invention the plants are mono and dicotyledonous plants, mosses or algae. An additional refinement according to the invention are transgenic plants as described above which contain a nucleic acid sequence or construct according to the invention or an expression cassette according to the invention. Furthermore, by derivatives we mean homologs of the sequences of the SEC. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487, for example, eukaryotic homologs, truncated sequences, single-strand DNA of the coding DNA sequence and without coding or RNA of the coding DNA sequence and without coding.
In addition, by homologs of the sequences of the SEC. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 means derivatives such as by way of promoter variants. These variants can be modified by one or more nucleotide exchanges, by insertion or insertions and / or elimination or deletions without, however, adversely affecting the functionality or efficiency of the promoters. In addition, promoters can have their efficiency increased by altering their sequence or being completely replaced by more effective promoters, even from foreign organisms. By derivatives it is also advantageously meant variants whose nucleotide sequence has been altered in the region from -1 to -2000 in front of the start codon in such a way that the gene expression and / or the expression of proteins is modified, preferably increasing . In addition, by derivatives it is also meant variants which have been modified at the 3 'end. Suitable promoters in the expression cassette are in principle promoters which can control the expression of foreign genes in organisms such as microorganisms such as protozoa such as ciliates, algae such as green, brown, red or blue algae such as Euglenia, bacteria such as gram-positive or gram-negative bacteria, yeasts such as Saccharomyces, Pichia or
Schizosaccharomyces or fungi such as Mortierella, Thraustochytrium or Schizochytrium or plants, advantageously in plants or fungi. The use is preferably made in particular of plant promoters or promoters derived from a plant virus. Advantageous regulation sequences for the method according to the invention are found, for example, in promoters such as eos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq-, T7, T5, T3 promoters, gal, tre, ara, SP6,? -PR or in? -PL which are advantageously employed in gram-negative bacteria. Other advantageous regulation sequences are found, for example, in the gram-positive promoters amy and SP02, in yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, EDH or in the promoters of plants, CaMV / 35S [Franck et al., Cell 21 (1980) 285-294], SSU, OCS, lib4, STLS1, B33, nos (= promoter of Nopaline Sintasa) or in the ubiquitin or phaseolin promoter. The expression cassette may also contain a chemically inducible promoter by means of which the expression of the exogenous sequences of SEQ. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 in organisms can be advantageously controlled in plants at particular time. Promoters of advantageous plants of this type are, by way of example, the PRP1 promoter [ard et al., Plant. Mol. Biol. 22 (1993), 361-366], a promoter inducible by benzenesulfonamide (EP 388 186), a promoter
tetracycline-inducible [Gatz et al., (1992) Plant J. 2,397-404], a salicylic acid-inducible promoter (O 95/19443), an abscisic acid-inducible promoter (EP 335 528) and an ethanol-inducible promoter or cyclohexanone (W093 / 21334). Other examples of plant promoters which can be advantageously used are the potato cytosolic FBPase promoter, the potato ST-SLI promoter (Stockhaus et al., EMBO J. 8 (1989), 2445-245), the phosphoribosil promoter. pyrophosphate amidotransferase of Glycine max (see also Accession number of the genetic bank U87999) or a specific promoter without diene as described in EP 249 676. Particularly advantageous are those plant promoters which assure the expression in tissues or parts / organs of plants in which the fatty acid biosynthesis or the precursor stages thereof, occurs, as in the endosperm or in the embryo of development for example. Particularly noteworthy are advantageous promoters which assure seed specific expression such as by way of example the USP promoter or derivatives thereof, the LEB4 promoter, the phaseolin promoter or the napin promoter. The particularly advantageous USP promoter cited according to the invention or its derivatives mediates very early gene expression in seed development [Baeumlein et al., Mol GenGenet, 1991, 225 (3): 459-67] -Other seed-specific promoters advantageous
which can be used for monocotyledonous or dicotyledonous plants are suitable promoters for dicotyledons such as promoters of the napin gene, likewise cited by way of example, of oilseed rape (US 5,608,152), the oleosin promoter from Arabidopsis (WO 98/45461 ), the phaseolin promoter from Phaseolus vulgaris (US 5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or the B4 promoter from legume (LeB4, Baeumlein et al., Plant J., 2, 2, 1992; 233 -239) or promoters suitable for monocotyledons such as the promoters of the Ipt2 or Iptl gene in barley (WO 95/15389 and WO 95/23230) or the promoters of the hordein gene of barley, the rice glutelin gene, the gene of rice orycin, the rice prolamin gene, the wheat gliadin gene, the white glutelin gene, the corn zein gene, the oat glutelin gene, the kasirin gene from sorghum or the secalin gene from rye which are described in WO 99/16890. Furthermore, particularly preferred are those promoters, which ensure expression in tissues, or parts of plants in which, for example, the biosynthesis of fatty acids, oils and lipids or the precursor stages thereof take place. Particularly however, they are promoters which ensure a specific expression of seeds. However, they are the promoters of the napin gene from oilseed rape (US 5,608,152), the USP promoter from Ficia
Faba (USP = protein from unknown seeds, Baeumlein et al., Mol.Gen Genet, 1991, 225 (3): 459-67), the promoter of the oleosin gene of. Arabidopsis (W098 / 45461), the phaseolin promoter (US 5,504,200) or the legumin B4 gene promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9). Other promoters to be mentioned are those of the Ipt2 or Iptl gene of barley (W095 / 15389 and WO95 / 23230) which mediate the specific expression of seed in monocotyledonous plants. Other promoters specific for advantageous seeds are promoters such as the rice, corn or wheat promoters described in WO 99/16890 or Amy32b, Amy6-6 or aleurain (SU 5,677,474), Be4 (rapeseed, US 5,530,149), glycinin (soybean, EP 571741), phosphenol pyruvate carboxylase (soybean, JP 06/62870), ADR12-2 (soybean, WO 98/0862), isocitratliase (rapeseed, US 5,689,040) or ß-amylase (barley, EP 781 849). As described above, the expression construct (= genetic construct, nucleic acid construct) may still contain other genes, which are to be introduced into organisms. These genes can be subject to separate regulation or be subjected to the same regulatory region as sequences of the SEC. FROM IDENT. DO NOT. : (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487. These genes are by way of example other biosynthesis genes, advantageously for fatty acid biosynthesis, vitamin biosynthesis, etc. , which allow increased synthesis.
In principle, all natural promoters with their regulatory sequences can be used as those named above for the expression cassette according to the invention and the method according to the invention. On top of this, synthetic promoters can also be used advantageously. In the preparation of an expression cassette several DNA fragments can be manipulated in order to obtain a nucleotide sequence, which reads practically in the correct direction and is equipped with a correct reading grid. To connect the DNA fragments (= nucleic acids according to the invention) from one adapter to another, they can bind to the fragments. The promoter and terminator regions can be usefully provided in the transcription direction with a linker or poly-linker containing one or more restriction sites for the insertion of this sequence. Generally, the linker has 1 to 10, mainly 1 to 8, preferably 2 to 6, restriction sites. In general, the size or linker within the regulatory region is less than 100 bp, often less than 60 bp, but at least 5 bp. The promoter can be either native or homologous as well as foreign or heterologous to the host organism, for example to the host plant. In the transcription direction 5 ', 3' the expression cassette contains the promoter, a sequence of
DNA which encodes the SEC gene. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and a region for transcription termination. Different termination regions can be exchanged with each other in any desired way. In addition, manipulations which provide adequate restriction interfaces or which remove excess DNA or restriction interfaces can be employed. In the. cases in which insertions, deletions or substitutions, such as transitions and transversions, come into consideration, in vitro mutagenesis, primer repair, restriction or ligation can be used. In suitable manipulations such as restriction, chewing or filling of protrusions for blunt ends, complementary ends of the fragments can be provided for ligation. For an advantageous high expression the binding of the ER retention signal specifies SEKDEL inter alia may be of importance (Schouten, A. et al., Plant Mol. Biol. 30 (1996), 781-792). In this way the average expression level is tripled or even quadrupled. Other retention signals which naturally occur in plant and animal proteins located in the ER can also be used for the construction of the cassette. In another preferred embodiment a plastidial target sequence is used as described
by Napier J.A. [Targeting of foreign proteins to the chloroplast, Methods Mol. Biol., 49, 1995: 369-376]. A preferred used vector comprising the target plastidial sequence is described by Colin Lazarus [Guerineau F., Woolston S., Brooks L., Mullineaux P. "An expression cassette for targeting foreing proteins into chloroplast; Nucleic Acids. Res., Dec. 9, 16 (23), 1988: 11380.] Preferred polyadenylation signals are plant polyadenylation signals, preferably those which substantially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J.3 (1984), 835 et seq.) Or corresponding functional equivalents An expression cassette is produced by fusion of a suitable promoter with suitable sequences of SEQ ID NO: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 together with the polyadenylation signal by common recombination and cloning techniques as described by example, in T. Maniatis, EF Frits ch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) as well as in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and
Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and iley-Interscience (1987). In the preparation of an expression cassette several DNA fragments can be manipulated to produce a nucleotide sequence which is usefully read in the correct direction and equipped with a correct reading grid. The adapters or linkers can be attached to the fragments to join the DNA fragments. The promoter and terminator regions can be usefully provided in the transcription direction with a linker or poly-linker containing one or more restriction sites for the insertion of this sequence. In general, the linker has 1 to 10, mainly 1 to 8, preferably 2 to 6, restriction sites. In general, the size of the linker within the regulatory region is less than 100 bp, frequently less than 60 bp, but at least 5 bp. The promoter can be either native or homologous as well as foreign or heterologous to the host organism, for example, to the host plant. In the direction of transcription 5 '-3' the expression cassette contains the promoter, a DNA sequence which encodes any gene of SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and a region for transcription termination. Different termination regions can be exchanged with each other in any desired way.
In the preparation of an expression cassette, several DNA fragments can be manipulated to produce a nucleotide sequence which usefully reads in the correct direction and is equipped with a correct reading grid. The adapters and linkers can be attached to the fragments to join the DNA fragments. The DNA sequences encoding the nucleic acid sequences used in the inventive processes such as the sequences of SEQ. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 contain all the sequence characteristics necessary to achieve the correct location of respective biosynthesis. Therefore, no target sequence is needed per se. However, such a location may be desirable and advantageous and therefore artificially modified or reinforced so that fusion constructions are also a preferred advantageous embodiment of the invention. Particularly preferred are the sequences which assure plastid targets. Under certain circumstances, the targets in other compartments (reported in: Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423) may also be desirable, for example, in vacuoles, the mitochondrion, the endoplasmic reticulum ( ER), peroxisomes, lipid structures or due to the lack of corresponding operative sequences in the compartment
of origin, the cytosol. As used herein, the term "environmental aggression" refers to any condition of sub-optimal growth and includes, but is not limited to, sub-optimal conditions associated with salinity, drought, temperature, metals, chemicals, pathogenic aggressions and oxidative, or combinations thereof. In the preferred embodiments, the environmental aggression may be by salinity, drought, heat or low temperature, or combinations thereof, and in particular may be by minimum water content or low temperature. Where the aggression by drought means any environmental aggression, which leads to a lack of water in plants or reduction of water supply to plants, where the aggression by low temperature means freezing of plants below + 4 ° C as well as cooling of plants below 15 ° C and where the aggression by high temperature means for example, a temperature above 35 ° C. The range of stress and response to aggression depends on the different plants which are used for the invention, ie, it differs for example between a plant such as wheat and a plant such as Arabidopsis. A common response of plants to environmental aggression is loss of yield or loss of quality. It will also be understood that as used in the specification and in the claims, "a" or "an" may
meaning one or more, depending on the context in which it is used. Thus, for example, the reference to "one cell" can mean that at least one cell can be used. As also used herein, the terms "nucleic acid" and "nucleic acid molecule" are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and DNA analogues. or RNA generated using nucleotide analogs. This term also encompasses the untranslated sequence located both at the 3 'and 5' ends of the coding region of the gene: at least about 1000 nucleotides of upstream sequence from the 5 'end of the coding region and at least about 200 nucleotides of downstream sequence from the 3 'end of the coding region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but preferably double-stranded DNA. An "isolated" nucleic acid molecule is one that is substantially separated from other nucleic acid molecules, which occur in the natural source of the nucleic acid. This means that other nucleic acid molecules are present in an amount less than 5% based on the weight of the amount of the desired nucleic acid, preferably less than 2% by weight, more preferably less
of 1% by weight, more preferably less than 0.5% by weight. Preferably, an "isolated" nucleic acid is free of some of the sequences that naturally flank the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the nucleic acid molecule encoding the stress related protein isolated contains less than about 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 is derived. In addition, an "isolated" nucleic acid molecule such as a cDNA molecule may be free of some other material with which it is naturally associated, or a culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when they are synthesized chemically. A nucleic acid molecule of the present invention, for example a nucleic acid molecule encoding an SRP or a portion thereof which confers tolerance and / or resistance to environmental aggression in plants, can be isolated using standard molecular biological techniques and the sequence information provided herein. For example, a cDNA can be isolated
encodes the Stress Related Protein of Arabidopsis thaliana from a cDNA library of a cDNA encoding Stress Related Protein from A. thaliana or Brassica napus, Glycine max, Zea mays or Oryza sativa can be isolated from from a cDNA library of Brassica napus, Glycine max, Zea mays or Oryza sativa respectively using all or a portion of one of the sequences of the SEC. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 and respectively. In addition, a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based on this sequence. For example, the mRNA can be isolated from plant cells (for example, by the guanidinium-thiocyanate extraction method of Chirgwin et al., 1979 Biochemistry 18: 5294-5299) and the cDNA can be prepared using reverse transcriptase (by example, Moloney MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based on one of the nucleotide sequences shown in SEQ. FROM IDENT. DO NOT.:
(4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487. A nucleic acid molecule of the invention can be amplified using cDNA or, alternatively genomic DNA, as a template and the primers of suitable oligonucleotides according to standard PCR amplification techniques. The nucleic acid molecule thus amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to a nucleotide sequence encoding SRP can be prepared by standard synthetic techniques, for example, using an automated DNA synthesizer. In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in the sequences of SEQ. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 that encodes the SRP (ie, "the coding region"), as well as 5 'untranslated sequences and 3 'untranslated sequences. In addition, the nucleic acid molecule of the invention can comprise only a portion of the coding region of one of the SEC nucleic acid sequences. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487, for example, a fragment which can be used as a probe or a primer or a fragment that encodes a portion biologically active of an SRP.
Portions of proteins encoded by the nucleic acid molecules encoding SRP of the invention are preferably biologically active portions described herein. As used herein, the term "biologically active portion of" an SRP is intended to include a portion, eg, a domain / motif, of a stress-related protein that participates in a tolerance response and / or resistance to Stress in a plant. To determine whether an SRP, or a biologically active portion of it, results in stress tolerance in a plant, a stress analysis of a plant comprising the SRP can be performed. Such methods of analysis are well known to those skilled in the art, as detailed in the Examples. More specifically, nucleic acid fragments encoding biologically active portions of an SRP can be prepared by isolating a portion of one of the SEC nucleic acid sequences. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 that expresses the encoded portion of the SRP or peptide (for example, by recombinant expression in vitro) and by evaluating the activity of the encoded portion of the SRP or peptide. The biologically active portions of an SRP are encompassed by the present invention and include peptides comprising amino acid sequences derived from the sequence
of amino acids of a gene encoding an SRP, or the amino acid sequence of a protein homologue to a SPR, which includes fewer amino acids than a full length SPR or the full length protein which is homologous to a SPR, and exhibits at least one enzymatic activity of a SPR. Typically, the biologically active portions (eg, peptides which are for example, 5, 10, 15, 20, 30, 35, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or reason with at least one activity of a SPR. In addition, other biologically active portions in which other regions of the protein are removed can be prepared by recombinant techniques and evaluated by one or more of the activities described herein. Preferably, the biologically active portions of an SRP include one or more domains / motifs selected from them that have biological activity. The term "biologically active portion" or "biological activity" means a SPR or a portion of a SPR which still has at least 10% or 20%, preferably 20%, 30%, 40% or 50%, especially preferably 60%, 70% or 80% of the enzymatic activity of the natural or starting enzyme. A nucleic acid molecule that encompasses a complete sequence of the nucleic acid molecules used in the process, for example, the polynucleotide of the invention, or a portion thereof, can be isolated
additionally by polymerase chain reaction, oligonucleotide primers based on this sequence or on parts thereof used. For example, a nucleic acid molecule comprising the entire sequence or part thereof can be isolated by polymerase chain reaction using oligonucleotide primers which have been generated at the base of this sequence - for example, the mRNA can be isolated from cells (e.g., by the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and the cDNA can be generated by reverse transcriptase (e.g., MLV Molon Reverse Transcriptase, available from Gibco / BRL, Bethesda, MD or AMV reverse transcriptase, obtainable from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for amplification, for example, as shown in Table 2, by means of polymerase chain reaction can be generated at the base of a sequence shown herein, for example, the sequence shown in SEQ. . FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 or the sequences derived from the polypeptides as shown in SEQ. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487. In addition, it is possible to identify conserved regions of several organisms by carrying out alignment of
sequence of proteins with the polypeptide used in the process of the invention, in particular with sequences of the polypeptide of the invention, from whose conserved regions, and in turn, primers. degenerates can be derived. The region conserved for the polypeptide of the invention is indicated in the alignment shown in the figure ALIGNMENT. The conserved regions are those, which show very little variation in the amino acid at a particular position of several homologs of different origin. The consensus sequences shown in Figure 2 are derived from such alignments. Degenerate primers can then be used by PCR for the amplification of novel protein fragments having aforementioned activity, for example, having a SPR activity of additional functional homologs of the polypeptide of the invention of other organisms. These fragments can then be used as a hybridization probe to isolate the entire genetic sequence. As an alternative, the absent 5 'and 3' sequences can be isolated by means of RACE-PCR (rapid amplification of cDNA ends). A nucleic acid molecule according to the invention can be amplified according to cDNA, or alternatively, genomic DNA as a template and suitable oligonucleotide primers,
following standard PCR amplification techniques. The nucleic acid molecule amplified in this way can be cloned into a suitable vector and characterized by means of a DNA sequence analysis. The 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. The nucleic acid molecules which are advantageously for the process according to the invention can be isolated on the basis of their homology to the nucleic acid molecules described herein using the sequences or part thereof as a hybridization probe and following the techniques of Hybridization standards under severe hybridization conditions. In this regard, 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 at least 15, 20 or 25 nucleotides in length which hybridize under severe conditions to the nucleic acid molecules described above, in particular those which encompass a nucleotide sequence of the nucleic acid molecule used in the process of the invention or which encodes a protein used in the invention or Nucleic acid molecule
the invention. Nucleic acid molecules with 30, 50, 100, 250 or more nucleotides can also be used. In addition to fragments of the SPR described herein, the present invention includes SRP analogs and analogs of natural origin and nucleic acids encoding SRP in a plant. "Homologs" are defined herein as two nucleic acids or proteins having similar nucleotide or amino acid sequences or "homologs", respectively. Homologs include allelic variants, orthologs, paralogs, agonists and SRP antagonists as defined below. The term "homologue" further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in the sequences with SEQ. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 (and portions thereof) due to the degeneracy of the genetic code and thus encodes the same SPR as that encoded by the amino acid sequences shown in the sequences with SEC. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487. As used herein a SRP "of natural origin" refers to an amino acid sequence of SRP that It happens naturally. The term "homology" means that the respective nucleic acid molecules or encoded proteins are functionally and / or structurally equivalent. The molecules of
Nucleic acids which are homologous to the nucleic acid molecules described above and which are derivatives of the nucleic acid molecules are for example, variations of the nucleic acid molecules which represent modifications that have the same biological function, in particular they encode proteins with the same or substantially the same biological function. These can be variations of natural origin, such as sequences of other varieties or species or mutations of plants. These mutations can be of natural origin or can be obtained by mutagenesis techniques. The allelic variations can be allelic variations of natural origin as well as variants produced synthetically or genetically designed. The equivalents structurally can for example, be identified by testing the binding of the polypeptide to antibodies or computer-based predictions. The equivalent structurally has the same immunological characteristic for example, which comprises similar epitopes. Functional equivalents derived from one of the polypeptides as shown in SEQ. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 according to the invention by substitution, insertion or removal have at least 30%, 35%, 40%, 45 % or 50%, preferably at least 55%, 60%, 65%, 70% or preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93%, 94% , very
especially preferably at least 95%, 98% or 99% homology with one of the polypeptides as shown in SEQ. FROM IDENT. DO NOT. : (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 according to the invention and distinguished by essentially the same properties as the polypeptide as shown in SEQ. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487. Functional equivalents derived from the nucleic acid sequence as shown in SEC. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 according to the invention by substitution, insertion or removal have at least 30%, 35%, 40%, 45 % or 50%, preferably at least 55%, 60%, 65% or 70% preferably 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. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487 according to the invention and encodes polypeptides having essentially the same properties as the polypeptide as shown in SEQ. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487. "Essentially the same properties" of a functional equivalent are understood above all as meaning that the functional equivalent has activity mentioned above, for example, conferring an increase in
fine chemical amount while increasing the amount of protein, the activity or function of the functional equivalent in an organism, for example, a microorganism, a plant or animal or plant tissue, plant or animal cells or a part thereof. By "hybridizing" is meant that such nucleic acid molecules hybridize under conventional hybridization conditions, preferably under severe conditions such as described for example, in Sambrook (Molecular Cloning; A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.-1-6.3.6. According to the invention, DNA molecules as well as RNA of the nucleic acid of the invention can be used as probes. In addition, as the template for the identification of Northern blot assays functional homologs as well as Southern blot assays can be performed. The Northern blot test advantageously provides additional information about the expressed gene product: for example, expression pattern, appearance of processing steps, such as splicing and capping, etc. The Southern blot assay provides additional information about the chromosomal location and
organization of the gene encoding the nucleic acid molecule of the invention. A preferred non-limiting example of severe hybridization conditions are hybridizations in sodium chloride / sodium citrate 6 x (= SSC) at about 45 ° C, followed by one or more washing steps in 0.2 x SSC, 0.1% SDS a 50 to 65 ° C, for example, at 50 ° C, 55 ° C or 60 ° C. The skilled artisan knows that these hybridization conditions differ as a function of the type of nucleic acid and, for example, when organic solvents are present, with respect to the temperature and concentration of the buffer. The temperature under "standard hybridization conditions" differ for example as a function of the nucleic acid type between 42 ° C and 58 ° C, preferably between 45 ° C and 50 ° C in an aqueous buffer with an O.lx concentration , 0.5x, lx, 2x, 3x, 4x or 5x SSC (pH 7.2). If the organic solvent or solvents are present in the aforementioned buffer, for example 50% formamide, the temperature under standard conditions is about 40 ° C, 42 ° C or 45 ° C. Hybridization conditions for hybrids of AD: DNA are for example, 0.1 x SSC and 20 ° C, 25 ° C, 30 ° C, 35 ° C, 40 ° C or 45 ° C, preferably between 30 ° C and 45 ° C. ° C. Hybridization conditions for hybrids of AD: RNA are preferably for example 0.1 x SSC and 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C or 55 ° C, preferably between 45 ° C and 55 ° C. The temperatures of
Hybridization mentioned above 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 qualified technician knows how to determine the required hybridization conditions with the help of textbooks, for example, those mentioned above, or 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. A further example of such a condition of severe hybridization is hybridization in 4XSSC at 65 ° C, followed by a wash in 0.1XSSC at 65 ° C for one hour. Alternatively, an exemplary severe hybridization condition is in 50% formamide, 4XSSC at 42 ° C. In addition, the conditions during the washing stage can be selected from the range of conditions defined by conditions of low severity (approximately 2X SSC at 50 ° C) and conditions of high severity (approximately 0.2X SSC at 50 ° C, preferably at 65 ° C) (20X SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). In addition, the temperature during the washing stage can rise from severe conditions
low at room temperature, approximately 22 ° C, at higher severity conditions at approximately 65 ° C. Both of the salt and temperature concentration parameters can be varied simultaneously, or even one of the two parameters can be kept constant while only the other is varied. The denaturants, for example, formamide or SDS, can also be used during hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C. Relevant factors such as i) treatment length, ii) salt conditions, iii) detergent conditions, iv) competing DNAs, v) temperature and vi) probe selection can be combined on a case-by-case basis so that not all the possibilities can be mentioned in the present. Thus, in a preferred embodiment, Northern blots are pre-hybridized with Rothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68 ° C for 2 hours. Hybridization with radioactive labeled probe is done overnight at 68 ° C. Subsequent washing steps are carried out at 68 ° C with lxSSC. For Southern blots the membrane is pre-hybridized with Rothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68 ° C for 2 hours. Hybridization with radioactive labeled probe is conducted overnight at 68 ° C. Subsequently, the hybridization buffer is removed and the
filter is washed in a short time using 2xSSC; 0.1% SDS. After removing the washing buffer, new 2xSSC buffer is added; 0.1% SDS and incubate at 68 ° C for 15 minutes. This washing step is carried out twice followed by an additional washing step using lxSSC; 0.1% SDS at 68 ° C for 10 minutes. Polypeptides having aforementioned activity, that is, conferring altered metabolic activity, derived from other organisms, can be encoded by other DNA sequences, which hybridize to the sequences shown in SEQ. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 under relaxed hybridization conditions and which encode the expression for peptides that confer an altered metabolic activity. In addition, some applications have to be carried out at low stringency hybridization conditions, without any consequence for the specificity of the hybridization. For example, a Southern blot analysis of total DNA could be examined with a nucleic acid molecule of the present invention and washed at low severity (55 ° C in 2xSPE0, 1% SDS). Hybridization analysis could reveal a simple pattern of only genes that encode polypeptides of the present invention or are used in the process of the invention, for example, having activity
mentioned herein to increase the fine chemical. A further example of such low stringency hybridization conditions is 4XSSC at 50 ° C or hybridization with 30 to 40% formamide at 42 ° C. Such molecules comprise those which are fragments, analogs or derivatives of the polypeptide of the invention or are used in the process of the invention and differ, for example, by way of elimination or deletions, insertion or insertions, substitution or substitutions, addition or additions and / or recombination or recombinations of amino acid and / or nucleotide or any other modifications known in the art either alone or in combination from the amino acid sequences described above or their or their underlying or underlying nucleotide sequences. However, it is preferred to use high stringency hybridization conditions. The 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, 80 bp, preferably at least 90, 100 or 110 pb. More preferably, they are fragments of at least 15, 20, 25 or 30 bp. Preferably, they are also hybridizations with at least 100 bp or 200 bp, most preferably at least 400 bp in length. In an especially preferred embodiment, the hybridization must be carried out with the complete nucleic acid sequence with conditions described above.
The terms "fragment", "fragment of a sequence" or "part of a sequence" mean a truncated sequence of the indicated original sequence. The truncated sequence (nucleic acid or protein sequence) can vary widely in length; the minimum size is a sequence of sufficient size to provide a sequence with at least one function and / or comparable activity of the indicated original sequence or by hybridizing with the nucleic acid molecule of the invention or used in the process of the invention under severe conditions , although the maximum size is not critical. In some applications, the maximum size is usually not substantially greater than that required to provide the desired activity and / or function (s) of the original sequence. In addition to fusion fragments and polypeptides of the SRPs described herein, the present invention includes homologs and analogs of naturally occurring SRPs and nucleic acids encoding SRP in a plant. "Homologs" are defined herein as two nucleic acids or polypeptides having similar or substantially identical nucleotide or amino acid sequences, respectively. Homologs include allelic variants, orthologs, paralogs, agonists and antagonists of the SRPs, as defined below. The term "homologous" covers
in addition nucleic acid molecules that differ from one of the nucleotide sequences shown in SEQ. FROM IDENT. NO .: (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487 (and portions thereof) due to the degeneracy of the genetic code and thus encodes the same SRP as that encoded by the nucleotide sequences shown in SEQ. FROM IDENT. DO NOT. : (4n + l) by n = 0 to 54 and (2n + l) by n = 110 to 487. As used herein, a "natural origin" SRP refers to an SRP amino acid sequence that occurs of natural form. Preferably, a SRP of natural origin comprises an amino acid sequence selected from the group consisting of polypeptides according to SEQ. FROM IDENT. NO .: (4n + 2) by n = 0 to 54 and (2n + 2) by n = 110 to 487. An SRP agonist can retain substantially the same or a subset of the biological activities of the SRP. An SRP antagonist can inhibit one or more of the activities of the naturally occurring form of the SRP. For example, the SRP antagonist can be competitively linked to a member downstream or upstream of the metabolic cascade of the cell membrane component that includes the SRP, or it binds to an SRP that mediates transport or compounds through such membranes, so that displacement is prevented from occurring. Nucleic acid molecules that correspond to natural and analogous allelic variants, orthologs and
Paralogs of an SRP cDNA can be isolated based on their identity to Saccharomyces cerevisiae, E. coli or based on their identity to the SRR nucleic acids of Brassica napus, Glycine max, Zea mays or Oryza sativa described herein using cDNAs of SRP or a portion thereof, as a hybridization probe according to standard hybridization techniques under severe hybridization conditions. In an alternative embodiment, the SRP homologs can be identified by selecting combinatorial libraries of mutants, eg, truncation mutants, from the SRP for SRP agonist or antagonist activity. In one embodiment, a varied library of SRP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a varied genetic library. A varied library of SRP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into genetic sequences, such that a degenerate set of potential SRP sequences is expressible as individual polypeptides, or alternatively as a set of larger fusion polypeptides. (e.g., for phage display) which contains the set of SRP sequences herein. There are a variety of methods that can be used to produce libraries of potential SRP homologs from a degenerate oligonucleotide sequence. The synthesis
Chemistry of a degenerate genetic sequence can be performed in an automatic DNA synthesizer, and the synthetic gene is then ligated into an appropriate expression vector. The use of a degenerate set of genes allows the provision, in a mixture, of all the sequences encoding the desired set of potential SRP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art. See, for example, Narang, S.A., 1983, Tetrahedron 39: 3; Itakura et al., 1984, Annu. Rev. Biochem. 53: 323; Itakura et al., 1984, Science 198: 1056; Ike et al., 1983, Nucleic Acid Res 11: 477. In addition, libraries or fragments of the SRP coding regions can be used to generate a varied population of SRP fragments for selection and subsequent selection of SRP homologs. In one embodiment, a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of an SRP coding sequence with a nuclease under conditions where breakage occurs only once per molecule, denaturing the double DNA strand, re-naturalizing DNA to form double-stranded DNA, which can include sense / antisense pairs from different broken products, removing portions of a single strand from reformed duplexes by treatment with nuclease SI, and binding the library of resulting fragment
in an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments and various sizes of the SRP. Several techniques are known in the art for selection of genetic products from combinatorial libraries made by point or truncation mutations, and for selection of cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid selection of genetic libraries generated by combinatorial mutagenesis of SRP homologs. The most widely used techniques, which are amenable to high throughput analysis, to select large genetic libraries, typically include cloning the genetic library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates the isolation of the vector encoding the gene whose product was detected. Recursive group mutagenesis (REM), a new technique that improves frequency or functional mutants in libraries, can be used in combination with screening trials to identify SRP homologues (Arkin and Yourvan, 1992, PNAS 89: 7811-7815; Delgrave et al., 1993, Polypeptide Engineering 6 (3): 327-331). In another modality, trials based on
cells can be harnessed to analyze a varied SRP library, using methods well known in the art. The present invention further provides a method for identifying a novel SRP, comprising (a) raising a specific antibody response to an SRP, or a fragment thereof, as described herein; (b) selecting the putative SRP material with the antibody, wherein the specific binding of the antibody to the material indicates the presence of a potentially novel SRP; and (c) analyzing the linked material in comparison to a known SRP, to determine its novelty. As stated above, the present invention includes SRPs and homologs thereof. To determine the percent sequence identity of two amino acid sequences (for example, one of the sequences according to SEQ ID NO: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487, and a mutant form thereof), the sequences are aligned for purposes of optimal comparison (for example, voids can be introduced into the sequence of one polypeptide for optimal alignment with the other polypeptide or nucleic acid) . The amino acid residues at corresponding amino acid positions are then compared. When a position in a sequence (for example, one of the sequences SEQ ID NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487) is occupied by the
same amino acid residue as the corresponding position in the other sequence (eg, a mutant form of the selected polypeptide sequence according to SEQ ID NO: (4n + 2) for n = 0 to 54 and ( 2n + 2) for n = 110 to 487, then the molecules are identical in that position.The same type of comparison can be made between two nucleic acid sequences.The percentage of sequence identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of sequence identity = numbers of identical positions / total numbers of positions x 100) Preferably, the isolated amino acid homologs included in the present invention are at least about 50-60% , preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90% or 90-95% and most preferably at least about 96 %, 97%, 98%, 99% or more id nticos a complete amino acid sequence according to SEQ. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487. In yet another embodiment, the isolated amino acid homologs included in the present invention are at least about 50-60% , preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and
greater preference at least about 96%, 97%, 98%, 99% or more identical to a complete amino acid sequence encoded by a nucleic acid sequence according to SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487. In other embodiments, the amino acid homologs of SRP have sequence identity over at least 15 contiguous amino acid residues, more preferably at least 25 contiguous amino acid residues and more preferably at least 35 contiguous amino acid residues according to SEC. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487. In another preferred embodiment, an isolated nucleic acid homologue of the invention comprises a nucleotide sequence which is less about 50-60%, preferably at least about 60-70%, more preferably at least about 70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence according to SEC. FROM IDENT. DO NOT. : (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 or to a portion comprising at least 20, 30, 40, 50, 60 consecutive nucleotides thereof. The preferable length of sequence comparison for nucleic acids is at least 75 nucleotides, more preferably at least 100 nucleotides and more preferably the full length of the region of
coding It is further preferred that the isolated nucleic acid homolog of the invention encodes an SRP, a portion thereof, which is at least 85% identical to an amino acid sequence according to SEQ. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487 and which functions as a modulator of a response to environmental aggression in a plant. In a more preferred embodiment, overexpression of the nucleic acid homologue in a plant increases the tolerance of the plant to environmental aggression. For the purposes of the invention, the percentage of sequence identity between two nucleic acid or polypeptide sequences is determined using the Vector NTI 6.0 (PC) software package (InforMax, 7600 isconsin Ave., Bethesda, MD 20814). A vacuum opening penalty of 15 and a vacuum extension penalty of 6.66 are used to determine the percent identity of two nucleic acids. A vacuum opening penalty of 10 and a vacuum extension penalty of 0.1 are used to determine the percent identity of two polypeptides. All other parameters are set in the preset parameters. For purposes of multiple alignment (Clustal W algorithm), the vacuum opening penalty is 10, and the empty extension penalty is 0.05 with matrix
blosum62. It will be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide is equivalent to a uracil nucleotide. In another aspect, the invention provides an isolated nucleic acid comprising a polynucleotide that hybridizes to the polynucleotide according to SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 under severe conditions. More particularly, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under severe conditions to the nucleic acid molecule comprising a nucleotide sequence according to SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length. Preferably, an isolated nucleic acid homolog of the invention comprises a nucleotide sequence which hybridizes under highly stringent conditions in the nucleotide sequence according to SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487, and it functions as a stress tolerance modulator in a plant. In a preferred embodiment, the overexpression of the nucleic acid homolog isolated in a plant increases a tolerance to the plant to environmental aggression. As used herein, with respect to the
hybridization for DNA to DNA stain, the term "severe conditions" refers in a modality to overnight hybridization at 60 ° C in 10X Denharts solution, 6X SSC, 0.5% SDS and 100 g / ml sperm DNA from denatured salmon. The spots are washed sequentially at 62 ° C for 30 minutes each time in 3X SSC / 0.1% SDS, followed by IX SSC / 0.1% SDS and finally 0. IX SSC / 0.1% SDS. As used herein also, "highly stringent conditions" refers to overnight hybridization at 65 ° C in 10X Denharts solution, 6X SSC, 0.5% SDS and 100 g / ml denatured salmon sperm DNA. . The spots are washed sequentially at 65 ° C for 30 minutes each time in 3X SSC / 0.1% SDS, followed by IX SSC / 0.1% SDS and finally 0. IX SSC / 0.1% SDS. Methods for nucleic acid hybridizations are described in Meinkoth and Wahl, 1984 Anal. Biochem. 138: 267-284; Ausubel et al. eds. , 1995, Current Protocols in Molecular Biology, chapter 2, Greene Publishing and Wiley-Interscience, New York; and Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part 1, Chapter 2, Elsevier, New York. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under severe or highly stringent conditions to a sequence of SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and
(2n + l) for n = 110 to 487 corresponds to a nucleic acid molecule of natural origin. As used herein, a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule having a naturally occurring nucleotide sequence (e.g., encodes a natural polypeptide). In one embodiment, the nucleic acid encodes an SRP of Saccharomyces cerevisiae, E. coli of natural origin or a SRP of Brassica napus, Glycine max, Zea mays or Oryza sativa of natural origin. By using the methods described above, and others known to those of skill in the art, one of ordinary skill in the art can isolate homologs from the SRPs comprising amino acid sequences shown in SEQ. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487. A subset of these homologs are allelic variants. As used herein, the term "allelic variant" refers to a nucleotide sequence that contains polymorphisms that leads to changes in the amino acid sequences of a SRP and that exists within a natural population (e.g., a species or variety of plant). Such natural allelic variations can normally result in 1-5% variance in an SRP nucleic acid. Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different plants, which can be carried
performed easily by using hybridization probes to identify the same genetic locus of SRP in those plants. Any and all variations of nucleic acid and polymorphisms or amino acid variations resulting in an SRP which are the result of natural allelic variation and which do not alter the functional activity of an SRP, are intended to be within the scope of the invention. An isolated nucleic acid molecule encoding an SRP having sequence identity with a polypeptide sequence of SEQ. FROM IDENT. NO .: (4n + 2) para.n = 0 to 54 and (2n + 2) for n = 110 to 487 may be created by introducing one or more nucleotide substitutions, additions or deletions in a SEC nucleotide sequence. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487, respectively, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded polypeptide. Mutations can be introduced in one of the sequences of the SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made in at least one or more predicted nonessential amino acid residues. 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 that have 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), unchanged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine) and side chains aromatics (for example, tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue provided in an SRP is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, the mutations may be randomly introduced throughout all or a portion of an SRP coding sequence, such as by saturation mutagenesis, and the resulting mutants may be selected for an SRP activity described herein for identify mutants that retain SRP activity. Following the mutagenesis of one of the sequences of the SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487, the encoded polypeptide can be expressed in a
Recombinant and polypeptide activity can be determined by analyzing the stress tolerance of a plant expressing the polypeptide as described herein. In addition, optimized SRP nucleic acids can be created. As used herein, "optimized" refers to a nucleic acid that is genetically engineered to increase its expression in a given plant or animal. To provide optimized SRP nucleic acids to the plant, the DNA sequence of the gene can be modified to 1) comprise preferred codons for highly expressed plant genes; 2) comprises an A + T content in base nucleotide composition to that found substantially in the plants; 3) forming a plant start sequence; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, RNA degradation and termination, or that form secondary structure hairpins or RNA splice sites. Increased expression of SRP nucleic acids in plants can be achieved by using the distribution frequency of codon usage in plants in general or a particular plant. Methods for optimizing nucleic acid expression in plants can be found in EPA 0359472; EPA 0385962; PCT Application No. WO 91/16432; U.S. Patent No. 5,380,831; U.S. Patent No. 5,436,391; Perlack et al., 1991, Proc. Nati Acad. Sci. USA 88: 3324-3328; and Murray et al., 1989, Nucleic Acids Res.
17: 477-498. As used herein, "preferred codon usage frequency" refers to the preference exhibited by a specific host cell in use of nucleotide codons to specify a given amino acid. To determine the frequency of use of a particular codon in a gene, the number of occurrences in that codon in the gene is divided by the total number of occurrences of all codons that specify the same amino acid in the gene. Similarly, the frequency of the preferred codon usage exhibited by a host cell can be calculated by averaging the frequency of use of the preferred codon in a large number of genes expressed by the host cell. It is preferable that this analysis be limited to genes that are highly expressed by the host cell. The percentage of deviation from the frequency of use of the preferred codon for a synthetic gene from that used by a host cell is first calculated by determining the percent deviation from the frequency of use of a single codon from that of the cell guest followed by obtaining the average deviation over all codons. As defined herein, this calculation includes unique codons (ie, ATG and TGG). In general terms, the total average deviation of the codon usage of an optimized gene from that of a host cell is calculated using the equation 1A = n = 1 Z Xn - Yn Xn times
100 Z where Xn = frequency of use for the codon in the host cell; Yn = frequency of use for codon n in the synthetic gene; n represents an individual codon that specifies an amino acid; and the total number of codons is Z. The total deviation of the codon usage frequency, A, for all amino acids should preferably be less than about 25% and more preferably less than about 10%. Therefore, an SRP nucleic acid can be optimized such that its distribution frequency of codon usage is, preferably, not deviated by more than 25% from that of highly expressed plant genes and, more preferably, not more than about 10%. In addition, consideration is given to the percentage content of G + C of the degenerate third base (monocotyledons appear to favor G + C in this position, while dicotyledons do not). It is also recognized that the XCG nucleotide (where X is A, T, C or G) is at least the preferred codon in dicots while the XTA codon is avoided in both monocotyledons and dicotyledons. The optimized SRP nucleic acids of this invention also preferably have GC and TA doublet evidence indices that closely approximate those of the chosen host plant. More preferably, these indices deviate from that of the host by no more than about 10-15%.
In addition to the nucleic acid molecules encoding the SRPs described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense to them. It is believed that antisense polynucleotides inhibit genetic expression of an objective polynucleotide by specifically binding the target polynucleotide and interfere with the transcription, splicing, transport, translation and / or stability of the target polynucleotide. The methods are described in the prior art to orient the antisense polynucleotide to chromosomal DNA, a primary RNA transcript, or a processed mRNA. Preferably, the target regions include splice sites, translation start codons, translation stop codons, and other sequences within the open reading frame. The term "antisense", for the purpose of the invention, refers to a nucleic acid comprising a polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcription, a processed mRNA, so as to interfere with the expression of the endogenous gene. "Complementary" polynucleotides are those that are capable of base pairing according to the Watson-Crick complementary rules of standards.
Specifically, purines with base pairs with pyrimidines to form a combination of guanine paired with cytosine
(G: C) and adenine paired with any thymine (A: T) in the case of DNA, or adenine paired with uracil (A: U) in the case of RNA. . It is understood that two polynucleotides can hybridize to each other even if they are not completely complementary to each other, with the proviso that each has at least one region that is substantially complementary to the other. The term "antisense nucleic acid" includes single-stranded RNA as well as double-stranded DNA expression cassettes that can be transcribed to produce antisense RNA. The "active" antisense nucleic acids are antisense RNA molecules that are capable of selectively hybridising with a primary transcription or mRNA encoding a polypeptide having at least 80% sequence identity with the SEC polypeptide. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487. The antisense nucleic acid can be complementary to a whole SRP coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an SRP. The term "coding region" refers to the region of the nucleotide sequence that comprises codons that are translated into amino acid residues. In another embodiment, the acid molecule
Antisense nucleic acid is antisense to a "non-coding region" of the coding strand of a nucleotide sequence that encodes an SRP. The term "uncoded region" refers to the 5 'and 3' sequences that flank the coding region that do not translate into amino acids (ie, they are also referred to as 5 'and 3' untranslated regions). The antisense nucleic acid molecule may be complementary to the entire coding region of SRP mRNA, but more preferably it is an oligonucleotide which is antisense to only a portion of the coding region or without coding of SRP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PKSRP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Typically, the antisense molecules of the present invention comprise an RNA having 60-100% sequence identity with at least 14 consecutive nucleotides of one of the SEC nucleic acid. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487. Preferably, the sequence identity will be at least 70%, more preferably at least 75%, 80% 85%, 90%, 95%, 98% and most preferably 99%. An antisense nucleic acid of the invention can be constructed using chemical synthesis and reactions of
enzymatic ligation using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using nucleotides of naturally occurring origin or differently modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the acids antisense and sense nucleics, for example, phosphorothioate derivatives and nucleotides substituted with acridine can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid, include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl -2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosilcuosine, inosine, β-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, β-adenine, 7-methylguanine, 5-methylamine-methyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl-quinosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil acid -5-oxyacetic acid (v), wibutoxosin, pseudouracil, cueosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,
4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector in which a nucleic acid has been subcloned in an antisense orientation (ie, RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a nucleic acid target of interest, also described in the following subsection). In yet another embodiment, the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, unlike the usual β units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids. Res. 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987). , FEBS Lett 215: 327-330). The antisense nucleic acid molecules of the invention are normally administered to a cell or generated in situ so that they hybridize with or bind to a cellular mRNA and / or genomic DNA encoding an SRP bywhich inhibits the expression of the polypeptide, for example, by inhibiting transcription and / or translation. Hybridization may be by conventional nucleotide complementarity to form a stable duplex, or for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the largest double helix groove . The antisense molecule can be modified so that it specifically binds to a receptor or an antigen expressed on a selected cell surface, for example, by binding the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor. or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, the vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral or eukaryotic promoter (including plant) are preferred. As an alternative to antisense polynucleotides, ribozymes, sense polynucleotides or double-stranded RNA (dsRNA) can be used to reduce the expression of an SRP polypeptide. By "ribozyme" is meant an enzyme based on catalytic RNA with ribonuclease activity the
which is capable of unfolding a nucleic acid of a single strand, such as an mRNA, to which it has a complementary region. Ribozymes (eg, hammerhead ribozymes described in Haselhoff and Gerlach, 1988, Nature 334: 585-591) can be used to catalytically unfold transcripts of SRP mRNA by inhibiting the translation of SRP mRNA. A ribozyme having specificity for a nucleic acid encoding SRP can be designed based on the nucleotide sequence of a SRP cDNA, as described herein (ie, SEQ ID NO: (4n + 1) for n = 0 to 54 and (2n + l) for n = 110 to 487) or on the basis of a heterologous sequence that is isolated according to methods taught in this invention. For example, a derivative of a Tetrahimena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence which is split into an mRNA encoding SRP. See, for example, U.S. Patent Nos. 4,987,071 and 5,116,742 to Cech et al. Alternatively, the SRP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a group of RNA molecules. See, for example, Bartel, D. and Szostak. J.W., 1993, Science 261: 1411-1418. In preferred embodiments, the ribozyme will contain a portion that is at least 7, 8, 9, 10, 12, 14, 16, 18 or 20 nucleotides, and most preferably 7 u
8 nucleotides, which have 100% complementarity to a portion of the target RNA. Methods for making ribozymes are known to those skilled in the art. See, for example, U.S. Patent Nos. 6,025,167; 5,773,260; and 5,496,698. The term "dsRNA", as used herein, refers to RNA hybrids comprising two strands of RNA. The ARNsds can be linear or circular in structure. In a preferred embodiment, the dsRNA is specific for a polynucleotide that encodes either the polypeptide according to SEQ. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487 or a polypeptide having at least 70% sequence identity with a polypeptide according to SEC. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487. The hybridizing RNAs can be substantially or completely complementary. By "substantially complementary" it is meant that when the two hybridizing RNAs are optimally aligned using the BLAST program as described above, the hybridizing portions are at least 95% complementary. Preferably, the dsRNAs will be at least 100 base pairs in length. Normally, the hybridizing RNAs will be of identical length without 5 'or 3' protruding and non-empty ends. However, dsRNAs have 5 'or 3' overhangs of up to 100 nucleotides can be used in the methods of the
invention The dsRNA may comprise ribonucleotides or ribonucleotide analogs, such as 2'-0-methyl ribosyl residues, or combinations thereof. See, for example, US Patent Nos. 4,130,641 and 4,024,222. A polydiboinosinic acid: polydibocitidylic acid dsRNA is described in US Pat. No. 4,283,393. Methods for making and using dsRNA are known in the art. One method comprises the simultaneous transcription of two complementary DNA strands, either in vivo, or in a single simple in vitro reaction mixture. See, for example, U.S. Patent No. 5,795,715. In one embodiment, dsRNA can be introduced into a plant or plant cell directly by standard transformation procedures. Alternatively, dsRNA can be expressed in a plant cell by transcribing two complementary RNAs. Other methods for the inhibition of endogenous gene expression, such as triple helix formation (Moser et al., 1987, Science 238: 645-650 and Cooney et al., 1988, Science 241: 456-459) and co-suppression ( Napoli et al., 1990, The Plant Cell 2: 279-289) are known in the art. Partial and total length cDNAs have been used for the co-suppression of endogenous plant genes. See, for example, U.S. Patent Nos. 4,801,340, 5,034,323, 5,231,020 and 5,283,184; Van der Kroll et al., 1990, The Plant
Cell 2: 291-299; Smith et al., 1990, Mol. Gen. Genetics 224: 477-481 and Napoli et al., 1990, The Plant Cell 2: 279-289. For sense deletion, it is believed that the introduction of a sense polynucleotide blocks the transcription of the corresponding target gene. The polynucleotide sense will have at least 65% sequence identity with the target plant gene or RNA. Preferably, the identity percentage is at least 80%, 90%, 95% or more. The polynucleotide sense introduced needs not to be of total length in relation to the target gene or transcription. Preferably, the sense polynucleotide will have at least 65% sequence identity with at least 100 consecutive nucleotides of one of the SEC nucleic acids. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487. The identity regions may comprise introns and / or exons and untranslated regions. The introduced polynucleotide sense can occur in the plant cell temporarily, or it can be stably integrated into a plant chromosome or extrachromosomal replicon. In addition, nucleic acid molecules encoding SRP from the same or other species such as SRP analogs, orthologs and paralogs, are intended to be within the scope of the present invention. As used herein, the term "analogs" refers to two nucleic acids that have the same or similar function,
but they have developed separately in unrelated organisms. As used herein, the term "orthologs" refers to two nucleic acids from different species that have been developed from a common ancestral gene by speciation. Normally, proteins that encode orthologs that have the same or similar functions. As also used herein, the term "paralogs" refers to two nucleic acids that are related by duplication within a genome. Paralogs usually have different functions, but these functions can be related (Tatusov, R.L. et al 1997 Science 278 (5338): 631-637). Analogs, orthologs and paralogs of a stress of natural origin related to protein may differ from the protein related to stress of natural origin by post-translational modifications, by amino acid sequence differences or by both. Post-translational modifications include in vivo and in vitro chemical derivatization of polypeptides, for example, acetylation, carboxylation, phosphorylation or glycosylation and such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modification enzymes. In particular, the orthologs of the invention will generally exhibit at least 80-85%, more preferably 90%, 91%, 92%, 93%, 94% and most preferably 95%, 96%, 97%, 98% or even 99% identity or homology with
all or part of a protein-related amino acid sequence related to stress of natural origin and exhibit a function similar to a protein related to stress. The orthologs of the present invention are also preferably able to participate in stress response in plants. In addition to naturally occurring variants of a stress-related protein sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation within a SEC nucleotide sequence. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487, thus leading to changes in the amino acid sequence of the protein related to encoded stress, without altering the Functional capacity of the protein related to stress or improving the functional capacity of the protein related to stress. For example, nucleotide substitutions that lead to amino acid substitutions in "non-essential" amino acid residues can be made in a sequence of SEQ. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487. Residue of "non-essential" amino acids is a residue that can be altered from the wild type sequence of a of the stress-related proteins without altering the activity thereof, while an "essential" amino acid residue is required for stress-related protein activity. Other amino acid residues, without
However (for example, those that are not conserved or only semi-conserved in the domain that has SRP activity) can not be essential for activity and thus are likely to be sensitive to alteration without altering the activity of the SRP. Accordingly, another aspect of the invention relates to nucleic acid molecules encoding stress-related proteins that contain changes in amino acid residues that are not essential for stress-related protein activity. Such SRP differs in amino acid sequence from a sequence of the SEC. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487, even retaining at least one of the stress-related protein activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence of SEQ. FROM IDENT. DO NOT. : (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to one of the Sequences of the SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487, more preferably at least approximately 60-70% homologous to one of the sequences of the SEC. FROM IDENT.
NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487, even more preferred to at least about 70-80%, 80-90%, most preferably 90% , 91%, 92%, 93%, 94% homologous to one of the SEC sequences. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and more preferably at least approximately 96%, 97%, 98% or 99% homologous to one of the sequences of the SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487. The preferred stress-related protein homologs of the present invention are preferably able to participate in the response to tolerance to stress in a plant. The homology (= identity) was calculated on the complete amino acid margin. The program used was PileUp (J. Mol.Evolution, 25 (1987), 351-360, Higgins et al., CABIOS, 5 1989: 151-153). The homologs of the sequences given in the SEC. FROM
IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 will be further understood as meaning, for example, homologs, analogs, orthologs and paralogs which have at least 30% of homology (= identity) at the level of amino acid derivative, preferably at least 50%, 60%, 70% or 80% homology, especially preferably at least 85% homology, most especially preferably 90%, 91 %, 92%, 93%, 94% homology, more preferably 95%, 96%, 97%, 98% or 99% homology. The homology (= identity) was calculated over the entire amino acid margin. The program
used was PileUp (J. Mol. Evolution., 25 (1987), 351-360, Higgens et al., CABIOS, 5 1989: 151-153) or the Gap and BestFit program [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970) and Smith and Waterman respectively (Adv. Appl. Math. 2; 482-489 (1981)] which are parts of the GCG software package [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991).] The aforementioned percentages of sequence homology are calculated with the BesFit or Gap program, preferably Gap, over the total sequence length with the following parameters used: Gap weight: 8, Weight of Length: 2. For the determination of the percentage of 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 example, the fasta software, which has now been used in the fasta 3 version (WR 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 FASTA Methods in Enzymology 183: 63-98). Another useful program for
calculation of homologies of different sequences is the standard blast program, which is included in the pending Biomax software (Biomax, Munich, Federal Republic of Germany). This unfortunately leads sometimes to sub-optimal results since blast does not always include complete sequences of the subject and the query. However, since this program is very efficient, it can be used for the comparison of a huge number of sequences. The following parameters are normally used for sequence comparisons: -p Program Name [String]; -d Database [String]; default = nr; -i Query File [File In]; default = stidin; -e Expectation value (E) [Real]; default = 10.0; -m alignment view options: 0 = peer formation; 1 = anchored query showing identities; 2 = no query-anchored identities; 3 = query-anchored flat, shows identities; 4 = consultation-anchored flat, without identities; 5 = consultation-anchored without identities and dull edges; 6 = query-anchored flat, without identities and dull edges; 7 = XML Blast output; 8 = tubular; 9 tubular with comment lines [Integer]; default = 0; -o BLAST report output file [File Out] Optional; default = stadout; -F Filter query sequence (DÜST with blastn, SEG with others) [String]; default = T; -G Cost to open a vacuum (zero makes the default behavior work) [Integer]; default = 0; -AND
Cost to extend a vacuum (zero makes the default behavior work) [Integer]; default = 0; -X X renounce value for empty alignment (in bits) (zero makes the default behavior work); blastn 30, megablst 20, tblastx 0, all the other 15 [Integer]; default = 0; -I Sample Gl's in defined lines [T / F]; default = F; -q Penalty for a nucleotide incompatibility (blastn only) [Integer]; default = -3; -r Reward for a nucleotide equivalent (blastn only) [Integer]; default = 1; -v Number of database sequences to show description only in line for (V) [Integer]; default = 500; -b Database sequence number to show alignments for (B) [Integer] by default = 250; -f Threshold to extend hits, by default if it is zero; blastp 11; blastn 0, blastx 12, tblastn 13; tblastx 13, megablast 0 [Integer]; default = 0; -g Perform vacuum alignment (not available with tblastx) [T / F] by default = T; -Q Genetic Query code for use [Integer]; default = 1; -D Genetic code DB (for tblastfnx] only) [Integer]; default = 1; -a Number of processors to use [Integer]; default = 1; -O SeqAlign file [File Out] Optional; -J Consider the query definition line [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 of zero for the actual size) [Actual]; default = 0; -K Number of best hits from a region to maintain (out by default, if a value of 100 is used is recommended) [Integer]; default = 0; -P 0 for multiple hits, 1 for single hits [Integer]; default = 0; -Y Effective length of the search space (use zero for the actual size) [Real]; default = 0; -S query threads for search against database (for blast [nx], and tablastx); 3 is both, 1 is superior, 2 is inferior [Integer]; default = 3; -T produces HTML output [T / F]; default = F; -I restricted database search for GI list [String] Optional; -U Low box usage when filtering the FASTA sequence [T / F] Optional; default = F; -and renounce value X for extensions without empty bits (0.0 makes the default behavior work); blastn 20, megablast 10; all the other 7 [Real]; default = 0.0; -Z renounce value X for final vacuum alignment in bits (0.0 operates the default behavior); blastn / megablst 50, tblastx 0, all the other 25 [Integer]; defect = 0; -R checkpoint file PSI-TBLASTN [File In] Optional; -n MegaBlast research [T / F]; default = F; -L location in query sequence [String] Optional; -A Multi-hit window size, by default if it is zero (blastn / megablast 0, all
the other 40 [Integer]; default = 0; -w Marco change penalty (OFF algorithm for blastx [Integer]; default = 0; -t Larger intron length allowed in tblastn to link HSPs (0 link disablements) [Integer], default = 0. high-quality results are achieved by using the algorithm of Needleman and Wunsch or Smith and Waterman, therefore, programs based on algorithms are preferred, and sequence comparisons can be made with the PileUp program (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5, 1989: 151-153) or preferably with the Gap and BestFit programs, 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 parts of the GCG software package [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991), Altschul et al. (1997) Nucleic Acids Res. 25: 3389 et seq.]. Preferably, the calculations to determine the percentages of sequence homology are made with the Gap program on the complete margin of the sequences. The following standard settings for the comparison of nucleic acid sequences were used: gap weight: 50, weight of length: 3, average equivalence: 10,000, average incompatibility:
0. 000. For comparison of amino acid sequences the same algorithms as described above or nucleic acid sequences can be used. High quality results are achieved by using the algorithm of Needleman and Wunsch or Smith and Waterman. Therefore, programs based on such algorithms are preferred. Advantageously, sequence comparisons can be made with the PileUp program (J. Mol.Evolution, 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or preferably with the Gap and BestFit programs. , 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, Wisconsin, USA 53711 (1991); Altschul et al. (1997) Nucleic Acids Res. 25: 3389 et seq. ] Therefore, preferably the calculations to determine the percentages of sequence homology are made with the Gap program on the complete margin of the sequences. The following standard settings for the comparison of amino acid sequences were used: gap weight: 8, weight of length: 2, average equivalence: 2,912, average incompatibility: -2,003. The variants will also cover, in particular,
functional variants which can be obtained from the sequence shown in SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 by means of elimination, insertion or substitution of nucleotides, the enzymatic activity of the derived synthetic proteins that are retained or improved . An isolated nucleic acid molecule encoding a stress-related protein homologous to a protein sequence according to SEQ. FROM IDENT. NO .: (4n + 2) for n = 0 to 54 and (2n + 2) for n = 110 to 487 can be created by introducing one or more nucleotide substitutions, additions or deletions in a SEC nucleotide sequence. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced in one of the sequences of the SEC. FROM IDENT. DO NOT. : (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Another route of enzyme mutagenesis, described in European Publication EP-A-0 909 821, is a method for using the Escherichia coli strain XLl-Red to generate mutants and alter the enzymatic activity. Preferably, conservative amino acid substitutions are made in one or more non-amino acid residues
planned essentials. 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 that have 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), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine) and side chains aromatics (for example, tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue provided in a stress related protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, the mutations can be randomly introduced along all or part of the sequence encoding the stress-related protein, such as by mutagenesis saturation, and the resulting mutants can be selected for an activity
of protein related to stress as described herein to identify mutants that retain protein activity related to stress or show protein activity related to improved stress. Following the mutagenesis of one of the nucleic acid sequences of the SEC. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487, the encoded protein can be expressed recombinantly and the activity of the protein can be determined by analyzing the stress tolerance of a plant that expresses the protein as described in the examples below. A useful method for determining the level of transcription of the gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for example, Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley, New York). This information at least partially demonstrates the degree of transcription of the gene. Total cellular RNA can be prepared from cells, tissues or organs by various methods, all well known in the art, such as that described in Bormann, E.R. et al., 1992 Mol. Microbiol, 6: 317-326. To evaluate the presence or relative amount of the translated protein from this mRNA, standard techniques, such as a Western blot, can be employed. These techniques are well known by someone of ordinary experiencein the art (see for example, Ausubel et al., 1988 Curren Protocols in Molecular Biology, Wiley, New York). The present invention also relates to a plant expression cassette comprising an SRP coding nucleic acid selected from the group comprising sequences of SEQ. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs or parts thereof operably linked to regulatory sequences and / or target sequences. In addition, the object of the invention is an expression vector comprising an SRP coding nucleic acid selected from the group comprising SEQ.NA. nucleic acid sequences. FROM IDENT. DO NOT. : (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs or parts thereof or a cassette for plant expression as described above, so the expression The nucleic acid encoding SRP in a host cell results in increased tolerance to environmental aggression when compared to a corresponding wild-type non-transformed host cell. The invention further provides an isolated recombinant expression vector comprising a stress-related protein encoding nucleic acid as described above, wherein the expression of the stress related vector or protein encoding nucleic acid,
respectively in a host cell results in tolerance and / or increased resistance to environmental aggression when compared to the corresponding non-transformed wild type of the host cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid" which refers to a circular double-stranded DNA loop within which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. In addition, types of vectors can be linearized nucleic acid sequences, such as transposons, which are pieces of DNA which can be copied or inserted by themselves. There have been 2 types of transposons found: simple transposons, known as Insertion Sequences and compound transposons, which can have several genes as well as the genes that are required for transposition. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and by
consequently, they are replicated together with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are frequently in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably since the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., retroviruses, adenoviruses and adeno-associated replication defective viruses), which perform equivalent functions. An expression cassette preferably contains regulatory sequences capable of driving the genetic expression in plant cells and operably linked so that each sequence can fulfill its function, for example, transcription termination by polyadenylation signals. The polyadenylation signals are those that originate from T-DNA of Agrobacterium tumefaciens such as gene 3 known as octopine synthase from the Ti plasmid pTiACH5 (Gielen et al., 1984 E BO J. 3: 835) or functional equivalents thereof. also all other functionally active terminators in plants are suitable.
Since the gene expression of the plant is not very frequently limited in translation levels, a plant expression cassette preferably contains other sequences operably linked as translation enhancers such as the overdirected sequence containing the 5'-untranslated leader sequence. from the tobacco mosaic virus that improves the protein by RNA ratio (Gallie et al., 1987 Nucí Acids Research 15: 8693-8711). The genetic expression of the plant must be operably linked to an appropriate promoter that confers genetic expression in a specific manner of appropriate cell or tissue. Promoters that drive constitutive expression (Benfey et al., 1989 EMBO J. 8: 2195-2202) are preferred as those derived from plant viruses such as 35S CaMV (Franck et al., 1980 Cell 21: 285-294), 19S CamV (see also US Patent No. 5352605 and PCT Application No. WO 8402913) or plant promoters such as those of a small Rubisco subunit described in US Patent No. 4,962,028. Additional advantageous regulatory sequences are included for example in plant promoters such as CamV / 35S [Franck et al., Cell 21 (1980) 285-294], PRPl [ard et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1, B33, LEB4, us or in the ubiquitin, napin or phaseolin promoter. Also advantageous in this connection are promoters
inducible agents such as the promoters described in EP-A-0 388 186 (inducible benzylsulfonamide), Plant. J. 2, 1992: 397-404 (Gatz et al., Inducible tetracycline), EP-A-0 335 528 (inducible abscisic acid) or WO 93/21334 (ethanol or inducible cyclohexanol). The promoters of additional useful plants are the cytosolic FBPase promoter or ST-LSI promoter of potato (Stockhaus et al., EMBO J. 8, 1989, 2445), the phosphoribosylphenophosphate amido transferase promoter from Glycine max (access no. from the gene bank U87999) or the specific promoter are described in EP-A-0 249 676. Additional particularly advantageous promoters are seed-specific promoters which can be used for monocotyledons or dicots and are described in US Pat. No. 5,608,152 (Napin seed promoter). rape), WO 98/45461 (phaseolin promoter from Arobidopsis), US 5,504,200 (phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica) and Baeumlein et al., Plant J ., 2, 2, 1992: 233-239 (LEB4 promoter from legumes). The promoters are useful in dicots. The following promoters are useful, for example, in the Ipt-2 or Ipt-1 promoter of monocotyledons from barley (WO 95/15389 and WO 95/23230) or hordein promoter from barley. Other useful promoters are described in WO 99/16890. It is possible in principle to use all
natural promoters with their regulatory sequences as those mentioned above for the novel process. In addition it is also possible and advantageous to use synthetic promoters. The genetic construct may further comprise genes which are to be inserted into the organisms and which are for example involved in resistance to stress. It is possible and advantageous to insert and express in host organisms regulatory genes such as genes for inductors, repressors or enzymes which are involved by their enzymatic activity in regulation, or one or more of all the genes of a biosynthetic path. These genes can be heterologous or homologous in origin. The inserted genes may have their own promoter or even be under the control of the same promoter as the SEC nucleic acid sequences. FROM IDENT. NO .: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 or their counterparts. The genetic construct advantageously comprises, for expression of the other genes present, additional 3 'and / or 5' terminal regulatory sequences that improve expression, which are selected for optimal expression depending on the selected host organism and gene or genes. These regulatory sequences are intended to make specific expression of the genes and the expression of the possible protein as mentioned above. This can
signifying, depending on the host organism, for example, that the gene is expressed or over-expressed only after induction, or that it is expressed immediately and / or over-expressed. The regulatory sequences or factors may also preferably have a beneficial effect on expression of the introduced genes, and thereby increase it. It is possible in this form for the regulatory elements to advantageously improve at the level of transcription using strong transcription signals such as promoters and / or enhancers. However, it is also possible to improve translation, for example, by improving the stability of the mRNA. Other preferred sequences for use in gene expression cassettes of plants are target sequences necessary to direct the gene product into its appropriate cell compartment (for review see Kermode, 1996 Crit. Rev. Plant Sci. 15 (): 285-423 and cited references in the present) such as the vacuole, the nucleus, all types of plastids such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, mitochondria, the endoplasmic reticulum, oil bodies, peroxis and other compartments of plant cells. Genetic expression of plants can also be facilitated by an inducible promoter (for review, see Gatz, 1997 Annu., Rev. Plant Physiol.
Mol. Biol. 48: 89-108). Chemically inducible promoters are especially suitable if gene expression is desired to occur in a specific manner of time. Table 1 lists several examples of promoters that can be used to regulate the transcription of stress-related protein nucleic acid coding sequences.
Table 1: Examples of stress-inducible tissue-specific promoters in plants
Other promoters, for example superpromotor (Ni et al., Plant Journal 7, 1995: 661-676), ubiquitin promoter (Callis et al., J. Biol. Chem., 1990, 265: 12486-12493; US 5,510,474; US 6,020,190; Kawalleck et al., Plant Molecular Biology, 1993, 21: 673-684) or 34S promoter (GenBank accession numbers M59930 and X16673) were similarly useful for the present invention and are known to a person skilled in the art. The technique. Preferred promoters of the developing stage are preferably expressed at certain stages of development. Preferred tissue and organ promoters include those that are preferably expressed in certain tissues or organs, such as leaves, roots, seeds or xylem. Examples of preferred tissue or organ-preferred promoters include, but are not limited to, preferred preferred fruit, preferred egg, male tissue preferred, preferred seed, preferred tugement, preferred tuber, preferred stem, preferred pericarp promoters , and leaf preferred, stigma preferred, pollen preferred, anther preferred, preferred of petal, preferred of sepals, preferred of pedicles, preferred of capsules, preferred of stems, preferred of root, and the like. Preferred seed promoters are preferably expressed during the development and / or germination of seeds. For example, preferred seed promoters
they may be preferred of embryo, preferred of endosperm, and preferred of seed husk. See Thompson et al., 1989, BioEssays 10: 108. Examples of preferred seed promoters include, but are not limited to cellulose synthase (celA), Ciml, gamma-zein, 1-globin-1, 19 kD corn zein (cZ19Bl) and the like. Other useful promoters in the expression cassettes of the invention include, but are not limited to, the higher bound chlorophyll a / b protein promoter, histone promoters, the Ap3 promoter, the β-conglycine promoter, the napin promoter, the promoter of soy lecithin, the 15kD corn zein promoter, the 22kD zein promoter, the 27kD zein promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2 and bronze promoters, the Zml3 promoter (US Patent No. 5,086,169), the corn polygalacturonase (PG) promoters (US Patent Nos. 5,412,085 and 5,545,546) and the SGB6 promoter (US Patent No. 5,470,359) as well as other synthetic or natural promoters. In addition, the flexibility to control heterologous gene expression in plants can be obtained by using the DNA binding domains and response elements from heterologous sources (i.e., DNA binding domains from different plant sources). An example of such a heterologous DNA binding domain is the domain of
LexA DNA link (Brent and Ptashne, 1985, Cell 43: 729-736). The invention further provides a recombinant expression vector comprising an SRP DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner that allows the expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to an SRP mRNA. Regulatory sequences that are operably linked to a nucleic acid molecule cloned in the antisense orientation which direct the continuous expression of the antisense RNA molecule in a variety of cell types can be chosen. For example, promoters and / or viral enhancers, or regulatory sequences which direct constitutive, tissue-specific or cell-type expression of the antisense RNA can be chosen. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or attenuated virus wherein the antisense nucleic acids are produced under the control of a high efficiency regulatory region. The activity of the regulatory region can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al., 1986, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in
Genetics, Vol. 1 (1), and Mol. et al., 1990, FEBS Letters 268: 427-430. Another aspect of the invention relates to host cells in which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchably herein. It is understood that such terms refer not only to the particular object cell but also apply to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to their mutation or environmental influences, such progeny can not in fact be identical to the progenitor cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, an SRP can be expressed in bacterial cells such as C. glutamicum, yeast, E. coli, insect cells, fungal cells or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae , ciliates, plant cells, fungi or other microorganisms such as C. glutamicum. Other suitable host cells are known to those skilled in the art. A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can
used to produce (ie express) an SRP. Accordingly, the invention further provides methods for producing SRPs using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (within which a recombinant expression vector encoding an SRP has been introduced, or within which genome a gene encoding a wild type SRP has been introduced or altered) in an appropriate medium until SRP occurs. In another embodiment, the method further comprises isolating the SRPs from the medium or the host cell. Another aspect of the invention relates to isolated SRPs, and biologically active portions thereof. An "isolated" or "purified" polypeptide or biologically active portion thereof is free of some of the cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes SRP preparations in which the polypeptide is separated from some of the cellular components of the cells in which they are produced naturally or recombinantly. In one embodiment, the language "substantially free of cellular material" includes preparations of an SRP having less than about 30% (by dry weight) of material without SRP (also referred to in
present as a "contaminating polypeptide"), more preferably less than about 20% material without SRP, even more preferably less than about 10% material without SRP and most preferably less than about 5% material without SRP. When the SRP or biologically active portion thereof is produced recombinantly, it is also preferably substantially free of the culture medium, ie, the culture medium represents less than about 20%, more preferably less than about 10%, and more preferably less than about 5% of the volume of the polypeptide preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of SRP in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of an SRP having less than about 30% (by dry weight) of chemical or chemical precursors without SRP, more preferably less than about 20% of chemical precursors or without chemicals without SRP, and more preferably less than about 10% chemical or chemical precursors without SRP, and more preferably less than about 5% chemical or chemical precursors without
SRP. In preferred embodiments, the isolated polypeptides or biologically active portions thereof, lack contaminating polypeptides from the same organism from which the SRP is derived. Typically, such polypeptides are produced by recombinant expression of, for example, an SRP in Saccharomyces cerevisiae, E. coli or Brassica napus, Glycine max, Zea mays or Oryza sativa in plants other than Saccharomyces cerevisiae, E. coli or microorganisms such as C glutamicum, ciliates, algae or fungi. The nucleic acid molecules, polypeptides, polypeptide homologs, fusion polypeptides, primers, vectors and host cells described herein can be used in one or more of the following methods: identification of Saccharomyces cerevisiae, E. coli or Brassica napus, Glycine max, Zea mays or Oryza sativa and related organisms; mapping genomes of organisms related to Saccharomyces cerevisiae, E. coli; identification and localization of Saccharomyces cerevisiae, E. coli or Brassica napus, Glycine max, Zea mays or Oryza sativa sequences of interest; evolutionary studies; determination of SRP regions required for function; modulation of an SRP activity; modulation of the metabolism of one or more cellular functions; modulation of the transmembrane transport of one or more compounds; modulation of
resistance to stress; and modulation of SRP nucleic acid expression. The SRP nucleic acid molecules of the invention are also useful for evolutionary and polypeptide structural studies. The metabolic and transport processes in which the molecules of the invention participate are used by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes of other organisms, the evolutionary relevance of the organisms can be evaluated. Similarly, such a comparison allows an evaluation of whose regions of the sequence are conserved and which are not, which may help to determine those regions of the polypeptide that are essential for the functioning of the enzyme. This type of determination is of value for polypeptide engineering studies and can give an indication of what the polypeptide can tolerate in terms of mutagenesis without loss of function. The manipulation of the SRP nucleic acid molecules of the invention can result in the production of SRPs having functional differences from the wild-type SRPs. These polypeptides can be improved in efficiency or activity, can occur in larger numbers in the cell than usual, or can be decreased in
efficiency or activity. There are a number of mechanisms by which the alteration of an SRP of the invention can directly affect the stress response and / or stress tolerance. In the case of plants that express SRPs, increased transport can lead to improved salt and / or solute division within the tissue and organs of the plant. By increasing the number or activity of transporter molecules that export ionic molecules from the cell, it may be possible to affect the saline tolerance of the cell. The effect of genetic modification on plants, C. glutamicum, fungi, algae or ciliates on stress tolerance can be assessed by cultivating the modified microorganism or plant under less than adequate conditions and then analyzing the growth and / or metabolism characteristics of the plant. plant. Such analysis techniques are well known to one skilled in the art, and include dry weight, wet weight, polypeptide synthesis, carbohydrate synthesis, lipid synthesis, evapotranspiration rates, plant yield and / or general culture, flowering, reproduction , seed settling, root growth, respiration rates, photosynthesis rates, etc. (Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular
Biology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III: Product recovery and purification, pages 469-714, VHC: einheim; Belter, P.A. et al., 1988, Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S., 1992, Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D., 1988, Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, pages 1-27, VCH: Weinheim; and Dechow, F.J. 1989, Separation and purification techniques in biotechnology, Noyes Publications). For example, yeast expression vectors comprising the nucleic acids described herein, or fragments thereof, can be constructed and transformed into Saccharomyces cerevisiae using standard protocols. The resulting transgenic cells can then be evaluated for failure or alteration of their tolerance to drought, salt and temperature stress. Similarly, plant expression vectors comprising the nucleic acids described herein, or fragments thereof, can be constructed and transformed into an appropriate plant cell such as Arabidopsis, soybean, rapeseed, corn, wheat, Medicago truncatula, etc., using similar protocols. The resulting transgenic cells and / or plants derived therefrom can then be evaluated
for failure or alteration of its tolerance to drought, salt, stress due to temperature and lodging. The engineering of one or more SRP genes of the invention may also result in SRPs having altered activities which directly impact the stress response and / or stress tolerance of algae, plants, ciliates or fungi, or other microorganisms such as C. glutamicum For example, normal biochemical processes of metabolism result in the production of a variety of products (for example, acid peroxide and other reactive oxygen species) which can actively interfere with these same metabolic processes. For example, peroxynitrite is known to nitrate tyrosine side chains, so some enzymes that have tyrosine in the active site are inactivated (Groves, JT, 1999, Curr Opin. Chem. Biol. 3 (2): 226- 235). Although these products are typically excreted, the cells can be genetically altered to transport more products that are typical for a wild-type cell. By optimizing the activity of one or more PKSRPs of the invention which are involved in the export of specific molecules, such as salt molecules, it may be possible to improve the stress tolerance of the cell. In addition, the sequences described herein, or fragments thereof, can be used to generate inactivated mutations in the genomes of various organisms,
such as bacteria, mammalian cells, yeast cells, and plant cells (Girke, T., 1998, The Plant Journal 15: 39-48). The resulting inactivated cells can then be evaluated for their ability or ability to tolerate various stress conditions, their response to various stress conditions, and the effect on the phenotype and / or genotype of the mutation. For other methods of genetic inactivation, see U.S. Patent No. 6,004,804, "Non-Chimeric Mutational Vectors" and Puttaraju et al., 1999, Spliceosome-mediated RNA trans-splicing as a tool for gene therapy, Nature Biotechnology 17: 246- 252. The aforementioned mutagenesis strategies for SRPs, which result in increased stress resistance do not mean that they are limiting; Variations in these strategies will be readily apparent to one skilled in the art. By using such strategies, and incorporating the mechanisms described herein, the nucleic acid and polypeptide molecules of the invention can be used to generate algae, ciliates, plants, fungi or other microorganisms such as C. glutamicum expressing mutated PKSRP nucleic acid and polypeptide molecules so that stress tolerance is improved. The present invention also provides antibodies that specifically bind to an SRP, or a portion thereof, as encoded by a nucleic acid.
described in the present. Antibodies can be made by many well-known methods (See for example, Harlow and Lane, "Antibodies; A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1988)). In brief, the purified antigen can be injected into an animal in an amount and at sufficient intervals to produce an immune response. The antibodies can either be purified directly or the spleen cells can be obtained from the animal. The cells can then be combined with an immortal cell line and selected for antibody secretion. The antibodies can be used to select nucleic acid clone libraries for cells that secrete the antigen. Those positive clones can then be sequenced. See, for example, Kelly et al., 1992, Bio / Technology 10: 163-167; Bebbington et al., 1992, Bio / Technology, 10: 169-175. The phrases "selectively link" and "specifically link" to the polypeptide refer to a binding reaction that is determinative of the presence of the polypeptide in a heterogeneous population of the polypeptides and other biologicals. Thus, under designated immunoassay conditions, the specific antibodies bound to a particular polypeptide do not bind in a remarkable amount to other polypeptides present in the sample. Selective binding of an antibody under such
conditions may require an antibody that is selected from its specificity for a particular polypeptide. A variety of immunoassay formats can be used to select antibodies that selectively bind to a particular polypeptide. For example, solid phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a polypeptide. See Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Publications, New York, (1988) for a description of immunoassay formats and conditions that could be used to determine selective binding. In some cases, it is desirable to prepare monoclonal antibodies from several hosts. A description of technique for preparing such monoclonal antibodies can be found in Stites et al., Eds. , "Basic and Clinical Immunology". (Lange Medical Publications, Los Altos, Calif., Fourth Edition) and references cited herein, and in Harlow and Lane, "Antibodies, A Laboratory Manual" Cold Spring Harbor Publications, New York (1988). Genetic expression in plants is regulated by the interaction of transcription factors of proteins with specific nucleotide sequences within the regulatory region of a gene. A common type of transcription factor contains zinc finger motifs (ZF). Each
ZF module is approximately 30 amino acids long folded around a zinc ion. The DNA recognition domain of a ZF protein is an a-helical structure that is inserted into the larger groove of the double DNA spiral. The module contains three amino acids that bind to the DNA with each amino acid by contacting a single base pair in the target DNA sequence. The ZF motifs are arranged in a modular repeat form to form a set of fingers that recognize a contiguous DNA sequence. For example, a three-finger ZF motif will recognize 9 bp of DNA. Hundreds of proteins have been shown to contain ZF motifs with between 2 and 37 ZF modules in each protein (Isalan M, et al., 1998 Biochemistry 37 (35): 12026-33; Moore M, et al., 2001 Proc. Nati, Acad. Sci. USA 98 (4): 1432-1436 and 1437-1441, US patents US 6007988 and US 6013453). The regulatory region of a plant gene contains many short DNA sequences (cis acting elements) that serve as recognition domains for transcription factors, including ZF proteins. Similar recognition domains in different genes allow the coordinated expression of several enzymes that encode genes in a metabolic pathway by common transcription factors. Variation in recognition domains between members of a genetic family facilitates differences in
genetic expression within the same genetic family, for example, between tissues and stages of development and in response to environmental conditions. Typical ZF proteins contain not only a DNA recognition domain but also a functional domain that allows the ZF protein to activate or repress the transcription of a specific gene. Experimentally, an activation domain has been used to activate transcription of the target gene (US Patent 5789538 and patent application W09519431), but it is also possible to bind a transcriptional repressor domain to ZF and thereby inhibit transcription (applications of patent WO00 / 47754 and WO2001002019). It has been reported that an enzymatic function such as nucleic acid cleavage can be linked to the ZF (patent application O00 / 20622). The invention provides a method that allows one skilled in the art to isolate the regulatory region of one or more stress-related protein coding genes from the genome of a plant cell to design the zinc finger transcription factors attached to a functional domain that will interact with the regulatory region of the gene. The interaction of the zinc finger protein with the plant gene can be designed in such a way as to alter the expression of the gene and preferably therefore alter the metabolic activity to confer tolerance
increased (or decreased) abiotic stress such as drought. The invention provides a method for producing a transgenic plant with a transgene encoding this designed transcription factor, or alternatively a natural transcription factor that modifies the transcription of the Stress Related Protein, particularly stress related protein gene to provide increased tolerance of environmental aggression. Such regulation of plant genes by artificial polydactyl zinc fingers has been demonstrated by Ordiz et al. (Regulation of transgene Expression in plants with polydactyl zinc finger transcription factors, Ordiz et al., PNAS, 99 (20) 13290-13295, 2002) or Guan et al. (Hertiable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors, PNAS, Vol. 99 (20), 13296-13301 (2002)). In particular, the invention provides a method for producing a transgenic plant with a nucleic acid encoding the stress-related protein, wherein the expression of the nucleic acid (s) in the plant results in increased tolerance to environmental aggression when compared to a wild-type plant, comprising: (a) transforming a plant cell with an expression vector comprising a nucleic acid encoding the stress-related protein, and (b) generating from a cell
vegetable a transgenic plant with an increased tolerance to environmental aggression when compared to a wild-type plant. For such plant transformation, binary vectors such as pBiNAr can be used (Hofgen and Willmitzer, 1990 Plant Science 66: 221-230). In addition, suitable binary vectors are for example, pBIN19, pBHOl, pGPTV or pPZP (Hajukiewicz, P. et al., 1994, Plant Mol. Biol., 25: 989-994). The construction of binary vectors can be performed by ligation of the cDNA into the T-DNA. 5 'to the cDNA a plant promoter activates transcription of the cDNA. A polyadenylation sequence is located 3 'to the cDNA. The tissue-specific expression can be achieved using a tissue-specific promoter as listed above. Also, any other promoter element can be used. For constitutive expression within the entire plant, the CaMV 35S promoter can be used. The expressed protein can be targeted to a cell compartment using a signal peptide, for example for plastids, mitochondria or endoplasmic reticulum (Kermode, 1996 Crit. Rev. Plant Sci. 4 (15): 285-423). The signal peptide is cloned 5 'in frame to the cDNA to achieve subcellular localization of the fusion protein. In addition, promoters that are sensitive to abiotic aggressions can be used with, such as the RD29A promoter from Arabidopsis. An expert in the art
it will recognize that the promoter used must be operably linked to the nucleic acid so that the promoter causes the transcription of the nucleic acid which results in the synthesis of an mRNA which encodes a polypeptide. Alternative methods of transfection include the direct transfer of DNA into developing flowers by electroporation or Agrobacterium-mediated gene transfer. The transformation of Agrobacterium-mediated plants can be carried out using, for example, GV3101 (pMP90) (Koncz and Schell, 1986 Mol.Gen.Genet., 204: 383-396) or strain of Agrobacterium tumefaciens LBA4404 (Ooms et al., Plasmid, 1982, 7: 15-29; Hoekema et al., Nature, 1983, 303: 179-180). The transformation can be carried out by standard transformation and regeneration techniques (Deblaere et al., 1994 Nucí Acids, Res 13: 4777-4788, Gelvin and Schilperoort, Plant Molecular Biology Manual, 2nd edition, Dordrecht: Kluwer Academic Publ. , 1995 - in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, BR and Thompson, JE, Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993. - 360 S ., ISBN 0-8493-5164-2). For example, rape seed can be transformed by transformation of cotyledons or hypocotyledons (Moloney et al., 1989 Plant Cell Reports 8: 238-242; De Block et al., 1989 Plant Physiol. 91: 694-701). The use of Agrobacterium antibiotics and selection of
plants depends on the binary vector and the Agrobacterium strain used for transformation. The selection of rape seed is usually done using kanamycin as a selectable plant marker. The gene transfer mediated by Agrobacterium to flax can be done using, for example, a technique described by Mlynarova et al., 1994 Plant Cell Report 13: 282-285. In addition, the transformation of soybeans can be carried out using, for example, a technique described in European Patent No. 0424 047, US Patent No. 5,322,783, European Patent No. 0397 687, US Patent No. 5,376,543 or US Patent No. 5,169,770. Corn transformation can be achieved by particle bombardment, incorporation of polyethylene glycol mediated DNA or by silicon carbide fiber technique (see for example, Freeling and Walbot "The corn handbook" Springer Verlag: New York (1993) ISBN 3- 540-97826-7). A specific example of corn transformation is found in U.S. Patent No. 5,990,387 and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256. The nucleic acid molecules encoding stress-related proteins of the invention have a variety of uses. Most notably, the nucleic acid and amino acid sequences of the present invention can be used to transform plant cells or
plants, which induces tolerance to stress such as drought, salinity and high cold. The present invention therefore provides a transgenic plant transformed by a nucleic acid encoding stress-related proteins (coding or antisense), wherein the expression of the nucleic acid sequence in the plant results in increased tolerance to environmental aggression when it is compared to a wild type plant. Increased stress tolerance is apparent as an increase in the yield or quality of the plant. The transgenic plant can be a monocotyledonous or dicotyledonous plant or a gymnosperm. The invention further facilitates that the transgenic plant may be selected from corn, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, borage, safflower, flaxseed, spring, rapeseed, canola and wild rape, cassava, pepper, sunflower, calendula, solanaceous plant such as potato, tobacco, eggplant and tomato, species Vicia, pea, alfalfa, thick plants such as coffee, cocoa, tea, Salix species, trees such as oil palm, coconut, perennial herbs, such as rye grass and pointer and forage crops, such as alfalfa and clover and Arabidopsis thaliana. In addition, the transgenic plant can be selected for example from spruce, pine or spruce. In particular, the present invention describes the use of the expression of proteins related to the
stress to design plants tolerant to drought, tolerant to salt and / or tolerant to the cold. This strategy has been demonstrated in the present for Arabidopsis thaliana, Ryegrass, Alfalfa, Rapeseed / Canola, Soybean, Corn and Wheat, but its application is not restricted to these plants. Accordingly, the invention provides a transgenic plant that contains a gene encoding stress related proteins selected from the SEC nucleic acid. FROM IDENT. DO NOT. : (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs of the aforementioned sequences, where the environmental aggression is drought, salt increased or temperature decreased or increased, but its application is not restricted to these adverse environments. Protection can be obtained against other adverse conditions such as, for example, heat, air pollution, heavy metals and chemical poisons. In the preferred modalities, environmental aggression is drought. The present invention also provides methods for modifying the stress tolerance of a plant comprising, modifying the expression of a gene encoding proteins related to stress in the plant. The invention provides that this method can be carried out in such a way that tolerance to stress increases. This can be done for example by the use of transcription factors. In particular, the present invention provides methods for
producing a transgenic plant that has an increased tolerance to environmental aggression when compared to a wild-type plant due to the increased expression of a protein related to stress in the plant. The growth of modified plants under stress conditions and then selecting and analyzing the growth characteristics and / or metabolic activity evaluates the effect of genetic modification on plants on tolerance and / or stress resistance. Such analysis techniques are well known to one skilled in the art. These include the following selection (Ropp Lexikon Biotechnologie, Stuttgart / New York: Georg Thieme Verlag 1992, "screening" p.701) of dry weight, wet weight, protein synthesis, carbohydrate synthesis, lipid synthesis, evapotranspiration rates, yield of plant and / or general crop, flowering, reproduction, seed settling, root growth, respiration rates, photosynthesis rates, etc. (Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol.17, Rehm et al., 1993 Biotechnology, vol.3, Chapter III: Product recovery and purification, pages 469-714, VCH: Weinheim; Belter , PA et al., 1988, Bioseparations: downstream processing for biotechnology, John Wiley and Sons, Kennedy, JF and Cabral, JMS, 1992, Recovery processes for biological materials, John Wiley and
Sons; Shaeiwitz, J.A. and Henry, J.D., 1988, Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, pages 1-27, VCH: Weinheim; and Dechow, F.J. 1989, Separation and purification techniques in biotechnology, Noyes Publications). The design of one or more genes encoding stress related proteins of the invention may also result in stress related proteins having altered activities which indirectly impact the stress response and / or stress tolerance of plants. For example, the normal biochemical processes of metabolism result in the production of a variety of products (for example, acid peroxide and other reactive oxygen species) which can actively interfere with these same metabolic processes (for example, peroxynitrite is known to it reacts with tyrosine side chains, so that some enzymes that have tyrosine are inactivated in the active site (Groves, JT, 1999 Curr Opin Opin Chem. Biol. 3 (2): 226-235). one or more proteins (enzymes) related to the stress of the invention, it may be possible to improve the stress tolerance of the cell Throughout this application, several publications were indicated Descriptions of all these publications and those references cited within those
publications in their totals are therefore incorporated for reference in this application in order to more fully describe the state of the art to which this invention pertains. It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes and variations may be made herein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not construed as limiting in any way. On the contrary, it will be clearly understood that various other embodiments, modifications and equivalents thereof, which after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and / or the scope of the claims. The invention also relates to the use of nucleic acid encoding an SRP selected from the group comprising the nucleic acid of the SEQ ID NO. NO: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs of the sequences mentioned above to prepare a plant cell with tolerance to increased environmental aggression. The sequences can also be used to prepare a plant with tolerance to increased environmental aggression. The object of the invention is also the use of
tolerance and / or increased resistance to environmental aggression and / or a nucleic acid encoding the SRP selected from the nucleic acid sequence group of the IDENT SEC. NO: (4n + l) for n = 0 to 54 and (2n + l) for n = 110 to 487 and / or homologs of the sequences mentioned above or parts thereof as markers for the selection of plants with increased tolerance to environmental aggression or as markers for the detection of stress in plants or plant cells.
Example 1 Design of stress-tolerant Arabidopsis plants by overexpressing stress-related protein genes.
Genetic cloning and transformation of Arabidopsis thaliana
Amplification The standard protocol for RNA extraction (Qiagen) and cDNA production (Invitrogen) is used for the RNA isolation procedure from different tissues of Brassica napus, Glycine max, Zea mays or Oryza sativa and the synthesis of its CDNA respective. The standard Pfu DNA polymerase protocol or a
Mixture of Pfu / Taq DNA polymerase (Herculase) was used for the amplification procedure. The amplified ORF fragments were analyzed by gel electrophoresis. Each primer consists of a universal 5 'end and 3' end specific ORF and therefore the universal sequences differ from the forward and reverse primers (the forward primer sequence contains an EcoRI for yeast or Smal for E. coli and the reverse primer sequence a Smal for yeast or SacI for restriction site E. coli) generally allowing unidirectional cloning success. Amplification using the Pfu or Herculase DNA polymerase protocol (Stratagene). Conditions: lx of PCR buffer, 0.2 mM dNTP, 100 ng of Saccharomyces cerevisiae genomic DNA (S288C) or 60ng of Escherichia coli K-12 genomic DNA (MG1655), 25 pmoles of forward primer, 25 pmoles of reverse primer, 2.5 u of DNA polymerase of Pfu or Herculase. 1st 3 'cycle for 2' yeast for E coli at 94 ° C, followed by 25 cycles for 30"at 94 ° C, 30" 55 ° C for yeast or 60 ° C for E. coli and 5-6 '72 ° C, followed by 1 cycle for 610 'to 72 ° C, final for 4 ° C to 8.
Table 2: Front and reverse priming sequences used for ORF amplification.
Preparation of vector The preferred binary vector lbxbigResgen for yeast and lbxSuperCoLic for E. coli which is based on the modified pPZP binary vector base (comprising the kanamycin gene for bacterial selection; Hajukiewicz, P. et al., 1994, Plant Mol. Biol., 25: 989-994) transported by selection bar gene marker (De Block et al., 1987, EMBO J. 6, 2513-2518) conducted by the masl 'promoter (Velten et al., 1984, EMBO J. 3, 2723-2730; Mentiste, Amedeo and Paszkowski, 1997, Plant J., 12, 945-948) in their T-DNA. In addition, the T-DNA contains the double strong 35S (Kay et al., 1987, Science 236, 1299-1302) for yeast or super-promoter (Ni et al., 1995, Plant Journal 7, 661-676) for E coli in front of a cloning cassette followed by the terminator nos (Depicker A. Stachel S. Dhaese P. Zambryski P. Goodman HMN. Nopalina synthase: tanscript mapping and DNA sequence, Journal of Molecular &Applied Genetics 1 (6): 561-73, 1982). The cloning cassette consists of the following sequence: 5'-TTG CTC TTC CAT GGC AAT GAT TAA TTA ACG AAG AGC
AA-3 '' Yeast: 5'-GGAATTCCAGCTGACCACCATGGCAATTCCCGGGGATC-3 or E. coli 5'-TTG CTC TTC CAT GGC AAT GAT TAA TTA ACG AAG AGC AA-3 ', respectively.
Other selection marker systems, such as the AHAS marker or other promoters, eg, super-promoter (see above), 35S promoter (see above), the ubiquitin promoter (Callis et al., J. Biol. Chem., 1990 , 265: 12486-12493, US 5,510,474, US 6,020,190, Kawalleck et al., Plant Molecular Biology, 1993, 21: 673-684) or 34S promoter (GenBank accession numbers M59930 and X16673) were similarly useful for present invention and are known to a person skilled in the art. The vector was linearized with EcoR and Smal for yeast or Smal and SacI for E. coli using the standard protocol provided by the supplier (MBI Fermentas, Germany) and purified using Qiagen columns (Qiagen, Hilden, Germany).
Ligation and transformation The fragments of ORF present (~ lOOng) were digested by EcoRI and Smal for yeast and Smal and SacI for E. coli using the standard protocol provided by the supplier (MBI Fermentas, Germany), purified using Qiagen columns (Qiagen, Hilden, Germany) and ligated into the cloning cassette of binary vector systems (~ 30ng) using standard procedures (Maniatis et al.). In the case of internal EcoRI, Smal and SacI restriction sites a blunt end cloning procedure was applied. The fragments of undigested ORF are
purified and ligated directly into the cloning cassette of the binary vector. In this case, the EcoRI site was filled by Klenow reaction and the SacI site was disaffiliated Pfu DNA polymerase. Ligation products were transformed into E. coli (DH5alpha) using a standard heat shock protocol (Maniatis et al.). The transformed colonies were cultured in an LB medium and selected by respective antibiotics (Km) for 16 hours at 37 ° C. Positive clones were identified by control PCR reactions using a combination of a specific vector and the respective ORF-specific primers.
Plasmid preparation Plasmid DNA was prepared from positive clones using standard protocols (Qiagen Hilden, Germany).
Transformation of Agrobacteria The plasmids were transformed into Agrobacterium tumefaciens (GV3101pMP90; Koncz and Schell, 1986, Mol.Gen.Genet., 204: 383-396) using heat shock or electroporation protocols. Transformed colonies were cultured in a YEP medium and selected by respective antibiotics (Rif / Gent / Km) for 2 days at 28 ° C. These
Agrobacterium cultures were used for the transformation of plants. The Arabidopsis thaliana was cultivated and transformed according to standard conditions Bechtold 1993 (Bechtold, N., Ellis, J. Pelletier, G. 1993. In the Agrobacterium plant the genetic transfer is mediated by infiltration of plants of Arabidopsis thaliana CR Acad. Sci. Paris 316: 1194-1199); Bent et al., 1994 (Bent, A., Kunkel, BN, Dahlbeck, D., Brown, KL, Schmidt, R., Giraudat, J., Leung, J., and Staskawicz, BJ 1994; PPCS2 of Arabidopsis thaliana : A leucine-rich repeat class of genes resistant to plant diseases: Science 265: 1856-1860). Transgenic A. thaliana plants were grown individually in pots containing a 4: 1 (v / v) mixture of earth and quartz sand in a York growth chamber. The standard growth conditions were: photoperiod of 16 hours of light and 8 hours of darkness, 20 ° C, 60% relative humidity, and a photon flux density of 150 μ ?. To induce germination, the sown seeds were kept at 4 ° C, in the dark, for 3 days. The plants were irrigated daily until they reached 3 weeks in which time the drought prevailed retaining water. In parallel, the relative humidity was reduced by 10% increments every second day to 20%. After approximately 12 days of retaining water, most of the
Plants showed visual symptoms of damage, such as wilting and rusting of the leaves, while tolerant plants were identified as visually turgid and green rozagante in color. The plants were classified for symptoms of drought damage compared to neighboring plants for 3 days in succession. Three successive experiments were conducted. In the first experiment, 10 independent T2 lines were seeded for each gene tested. The percentage of plants that does not show visual symptoms of damage was determined. In the second experiment, the lines that had been classified as tolerant in the first experiment were placed through a confirmation screen according to the same experimental procedures. In this experiment, the plants of each tolerant line were cultured and treated as above. In the third experiment, at least 7 copies of the most tolerant line were cultured and treated as above. The average and maximum number of drought survival days after the wild type control that had died visually were determined. In addition, chlorophyll fluorescence measurements were measured in stressed and non-stressed plants using a Mini-PAM (Heinz Walz GmbH, Effeltrich, Germany). In the first experiment, after 12 days of drought, the Arabidopsis thaliana non-transgenic control and the
More transgenic lines expressing other transgenes in the test showed extreme visual symptoms of stress including necrosis and cell death. Several plants that express the genes retained viability as shown by their turgid appearance and green maintenance. The second experiment was compared to a smaller number of independent transgenic lines for each gene, but a greater number of progeny within each independent transformation event. This experiment confirmed the previous results. Those lines containing the yeast genes encoding specific SRP survived longer than the controls. In some cases the transgenic line survived more than 3 days after the controls had died. According to the results of the first and second experiments some major lines containing the yeast genes encoding SRP-specific were identified, which showed the best results with respect to the average survival days after the wild type and / or the percentage of hits In a third experiment, these major lines were tested with multiple copies (4-80 plants per line). The average number of survival days of longer line plants longer than wild type was measured. That is, the number 1 'means that, on average, the plants that over-
They express this ORF, on average they survived 1 day longer than the wild type. The value for T in this column is '0'. The results are summarized in Table 3. Table 3: Drought tolerance of transgenic Arabidopsis thaliana expressing the various SRP coding genes from Saccharomyces cerevisiae or E. coli after the imposition of stress to drought in plants of 3 weeks in a third experiment using several plants of a transgenic line (experiment 3). Drought tolerance is measured by the indicated number of transgenic plants (Plants tested) as the average number of days (average days of survival after WT) that the transgenic plants survived after control (non-transformed wild type). For WT, this column has the value 0 '.
In a further experiment, for individual major lines, other lines containing the same
genetic construction, but resulting from a different transformation event were tested. In three lines, the specific yeast coding genes of SRP are incorporated at a different site in the genome of the plant. The results are summarized in table 4 according to table 3. The results demonstrate the dependence of tolerance and / or resistance to stress in plants in the expression of SRP, instead of the insertion event. Table 4: Drought tolerance of transgenic Arabidopsis thaliana expressing genes encoding SRPs selected from Saccharomyces cerevisiae or E. coli after imposing stress to dryness in 3-week plants in a third experiment using a multi-line transgenic plant independent each (experiment 3). Drought tolerance is measured by the indicated number of transgenic plants (Plants tested) as the average number of days (Average days of survival after WT) than the transgenic plants that survive after control (wild type not transformed). For WT, this column has the value? 0 '.
Chlorophyll fluorescence measurements of photosynthetic performance confirmed that severe stress to the
drought completely inhibited photosynthesis in control plants, but larger transgenic lines maintained the photosynthetic function for longer (Table 5). Table 5: Drought tolerance of transgenic Arabidopsis thaliana expressing the various SRP-encoding genes from Saccharomyces cerevisiae or E. coli after the imposition of drought stress on 3-week plants, in a third experiment using several plants of a transgenic line (experiment 3). Drought tolerance is reported as photosynthetic performance measured by chlorophyll fluorescence measured at three different time points during the drought stress experiment, and compared to non-transformed wild type control. For each transgenic line, the average of 5 copies of plants is indicated, the value of wild type is the average of 20-25 plants measured in the same experiment.
Example 2 Design stress-tolerant Arabidopsis plants by over-expressing genes encoding stress-related proteins from Saccharomyces cereviesae or E. coli using stress-inducible and tissue-specific promoters. Transgenic Arabidopsis plants were created as in example 1 to express the transgenes encoding stress-related proteins under the control of any tissue-specific or stress-inducible promoter. The constitutive expression of a transgene can cause harmful side effects. Stress inducible expression was achieved using promoters selected from those listed above in Table 1. T2 generation plants with drought stress were produced and treated in two experiments. For the first drought experiment, the plants were stripped of water until the plant and the soil were dehydrated. Several times after water retention, a normal watering schedule was summed up and the plants were grown to mature. The yield of seeds was determined as seeds per plant. In an equivalent degree of stress to drought, tolerant plants were able to resume normal growth and produce more seeds than control plants.
transgenic The proline content of the leaves and the opening of the stoma were also measured several times during drought stress. The tolerant plants maintained a lower proline content and a larger stoma opening than the non-transgenic control plants. An alternative method to impose water stress on transgenic plants was by treatment with water containing an osmolyte such as polyethylene glycol (PEG) at specific aqueous potential. Since PEG can be toxic, plants were only given a short-term exposure and then normal irrigation was summarized. As in the above, the seed yields were measured from mature plants. The response was measured during the stress period by physical measurements, such as stoma opening or osmotic potential, or biochemical measurements, such as proline accumulation. Tolerant plants had high seed yields, maintained their stoma opening and showed only slight changes in osmotic potential and proline levels, while susceptible non-transgenic control plants closed their stoma and exhibited increased osmotic potential and proline levels. Transgenic plants with a constitutive promoter that controls transcription of the transgene were compared in those plants with an inducible promoter by
drought in the absence of stress. The results indicate that the metabolite and gene expression changes did not occur when plants with the inducible stress promoter were cultured in the absence of stress. These plants also had higher seed yields than those with the constitutive promoter. Example 3 Overexpression of genes related to stress from Saccharomyces cerevisiae or E. coli facilitates tolerance to multiple abiotic aggressions. Plants that exhibit tolerance of abiotic aggression often exhibit tolerance to environmental aggression or to a herbicide that generates oxygen free radicals. This phenomenon of cross-tolerance is not understood on a mechanistic level (McKersie and Leshem, 1994). However, it is reasonable to expect that plants exhibiting improved tolerance to drought due to the expression of a transgene may also exhibit tolerance to low temperatures, freezing, salt, airborne contaminants such as ozone, and other abiotic aggressions. In support of this hypothesis, the expression of several genes is over- or un-regulated by multiple factors of abiotic aggression including cold, salt, osmosis, ABA, etc. (eg, Hong et al. (1992) Developmental and organic- specific expression of an ABA- and stress-induced protein in barley Plant Mol Biol 18: 663-674;
Jagendorf and Takabe (2001) Inducers of glycinebetaine synthesis in barley. Plant Physiol 127: 1827-1835); Mizoguchi et al. (1996) A gene encoding a mitogen-activated protein kinase and a S6 ribosomal protein kinase by contact stress, cold and water in Arabidopsis thaliana. Proc Nati Acad Sci U S A 93: 765-769; Zhu (2001) Cell signaling under salt, water and cold stresses. Curr Opin Plant Biol 4: 401-406). To determine salt tolerance, seeds of Arabidopsis thaliana (100% decolorizing agent, 0.1% TritonX for five minutes, twice and rinsed five times with ddH20) were sterilized. The seeds were plated in an unselected medium (1/2 MS, 0.6% phytagar, 0.5 g / L MES, 1% sucrose, 2 g / ml benamilo). The seeds were allowed to germinate for approximately ten days. In the 4-5 stage of the leaf, the transgenic plants were planted in pots of 5.5 cm in diameter and allowed to grow (22 ° C, continuous light) for approximately seven days, irrigating them when necessary. To start the assay, two liters of 100 mM NaCl and 1/8 MS were added to the tray under the pots. To the tray containing the control plants, three liters of 1/8 MS were added. The NaCl supplement concentrations were increased in stages by 50 mM every 4 days up to 200 mM. After saline treatment with 200 mM, the weights
Fresh and dried plants as well as seed yields were determined. To determine the tolerance to cold, seeds of the transgenic and cold lines were germinated and cultivated for approximately 10 days in the 4-5 stage of the leaf as in the previous. The plants were then transferred to cold temperature (5 ° C) and cultivated by stages of flowering and settling of developmental seeds. Photosynthesis was measured using chlorophyll fluorescence as an indicator of photosynthetic integrity and photosystem integrity. The yield of seeds and the dry weight of the plants were measured as an indicator of biomass production in plants. Plants that had tolerance to salinity or cold had higher yield of seeds, photosynthesis and production of dry matter than susceptible plants. EXAMPLE 4 Design of stress-tolerant alfalfa plants by over-expressing genes related to aggression from Saccharomyces cerevisiae or E. coli A clone of regeneration of alfalfa (Medicago sativa) was transformed using the method of (cKersie et al., 1999 Plant Physiol 119: 839-847). The regeneration and transformation of alfalfa is a dependent genotype and therefore
both a regeneration plant is required. Methods for obtaining regeneration plants have been described. For example, these may be selected from the Rangelander variety (Agriculture Canada) or any other variety of commercial alfalfa as described by Brown DCW 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 (Alker et al., 1978 Ann J Bot 65: 654-659). Petiole explant was co-cultured 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 (eg, An, G. in Agrobacterium Protocols, Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, New Sweater) . Many are based on the vector pBIN19 described by Bevan (Nucleic Acid Research, 1984. 12: 8711-8721) which includes a cassette of gene expression of the plant flanked by the left and right margin sequences from the Ti plasmid of Agrobacterium tumefaciens . A gene expression cassette of the plant consists of at least two genes - a selection marker gene and a plant promoter that regulates the transcription of the genomic cDNA or genomic DNA
characteristic. Several selection marker genes can be used, including the Arabidopsis gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Patent Nos. 57673666 and 6225105). Similarly, several promoters can be used to regulate the characteristic gene that provides constitutive, developmental, tissue or environmental regulation of genetic transcription. In this example, the 34S promoter (Accession number GenBank M59930 and X16673) was used to provide constitutive expression of the characteristic gene. The explants were co-cultured for 3 days in the dark in an SH induction medium containing 288 mg / L of Pro, 53 mg / L of thioproline, 4.35 g / L of K2S04, and 100 μg of acetosyringinone. The explants were washed in medium-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 the growth of Agrobacterium After several weeks, the somatic embryos were transferred to a BOÍ2Y development medium containing no growth regulators, no antibiotics, and 50 g / L of sucrose. The somatic embryos were subsequently germinated in medium-strength Murashige-Skoog medium. Rooted seedlings were transplanted in pots and grown in a greenhouse.
Transgenic TO plants were propagated by protrusion clippings and rooted in a Turface growth medium. The plants were defoliated and cultured at a height of approximately 10 cm (approximately 2 weeks after defoliation). The plants were then subjected to drought aggression in two experiments. For the first drought experiment, the seedlings received no water for a period of up to 3 weeks at which time the plant and soil were dried. Several times after water retention, a normal watering schedule was resumed. One week after resuming the risk, the fresh and dry weights of the outbreaks were determined. To an equivalent degree of drought aggression, tolerant plants were able to resume normal growth while susceptible plants had died or suffered noticeable damage resulting in less dry matter. The proline content of the leaves and the opening of the stoma were also measured several times during the drought aggression. The tolerant plants maintained a lower proline content and a larger stoma opening than the non-transgenic control plants. An alternative method for imposing aqueous aggression on the transgenic plants was by treatment with a solution in specific aqueous potential, which contains an osmolyte such as polyethylene glycol (PEG). The treatment with
PEG was given to any detached leaves (eg, Djilianov et al., 1997 Plant Science 129: 147-156) or roots (Wakabayashi et al., 1997 Plant Physiol 113: 967-973). Since PEG can be toxic, plants were only given a short-term exposure. The response was measured as physical measurements such as stomatal opening or osmotic potential, or biochemical measurements such as proline accumulation. Tolerant plants maintained their stoma opening and showed only slight changes in osmotic potential, while susceptible non-transgenic control plants closed their stoma and exhibited increased osmotic potential. In addition, changes in proline and other metabolites were lower in tolerant transgenic plants than in non-transgenic control plants. The tolerance of salinity and cold were measured using methods as described in example 3. Plants that had tolerance to salinity or cold had higher yields of seeds, photosynthesis and production of dry matter than susceptible plants. Example 5 Design of stress-tolerant weed plants by over-expressing genes related to aggression from Saccharomyces cerevisiae or E. coli Seeds of different varieties of weeds
They can be used as explant sources for transformation, including the commercial variety Gunne available from the seed company Svalof eibull of the variety Affinity. Seeds were surface sterilized sequentially with 1% Tween-20 for 1 minute, 100% decolorizing agent for 60 minutes, 3 rinses with 5 minutes each with de-ionized and distilled H20, and then germinated for 3- 4 days in humidity, the sterile filter paper in the dark. The seedlings were further sterilized for 1 minute with 1% Tween-20, 5 minutes with 75% decolorizing agent, and rinsed 3 times with ddH20, 5 minutes each. Superficially sterilized seeds were placed in the callus induction medium containing basal salts and Murashige and Skoog vitamins, 20 g / 1 sucrose, 150 mg / 1 asparagine, 500 mg / 1 casein hydrolyzate, 3 g / 1 Phytagel, 10 mg / 1 BAP, and 5 mg / 1 dicamba. Plates were incubated in the dark at 25 ° C for 4 weeks for seed germination and induction of embryogenic callus. After 4 weeks in the callus induction medium, the shoots and roots of the seedlings were cut, the callus was transferred to fresh medium, kept in culture for another 4 weeks, and then transferred to an MSO medium in the light for 2 weeks. Various pieces of calluses (11-17
weeks) were screened through a 10 mesh screen and placed in a callus induction medium, or were cultured in 100 ml of a liquid tart callus induction medium (same medium as for callus induction with agar) in a 250 ml flask. The flask was wrapped in aluminum foil and stirred at 175 rpm in the dark at 23C for 1 week. The liquid culture was sieved with a 40 mesh screen that harvests the cells. The fraction collected in the sieve was plated and cultured in a medium of solid tares callus induction for 1 week in the dark at 25 ° C. The callus was then transferred to and cultured in an MS medium containing 1% sucrose for 2 weeks. The transformation can be achieved with any Agrobacterium with particle bombardment methods. An expression vector is created containing a constitutive plant promoter and the gene cDNA in a pUC vector. Plasmid DNA was prepared from E. coli cells using the Qiagen kit according to the manufacturer's instruction. Approximately 2 g of the embryogenic cells were scattered in the center of a sterile filter paper in a Petri dish. An aliquot of liquid MSO with 10 g / 1 of sucrose was added to the filter paper. The gold particles (1.0 μ size) were coated with plasmid DNA according to the method of Sanford et al., 1993 and were delivered to the embryogenic callus with the following parameters: 500 μ?
of particles and 2 pg of DNA per stem, 1300 psi and a target distance of 8.5 cm from the stop plate to the callus plate and 1 stem per callus plate. After the bombardment, the calluses were transferred back to the fresh callus development medium and kept in the dark at room temperature for a period of 1 week. The callus was then transferred to growth conditions in the light at 25 ° C to initiate embryonic differentiation with the appropriate selection agent, for example 250 nM of Arsenal, 5 mg / 1 of PPT or 50 mg / L of kanamycin. The shoots resistant to the selection agent sprouted and rotted once they were transferred to the ground. 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 the DNA was electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The DIG Probé Synthesis PCR Kit (Roche Diagnostics) is used to prepare a probe labeled with digoxigenin by PCR, and is used as recommended by the manufacturer. Vegetative T0 weed plants were propagated vegetatively by removing stems. The transplanted stems were kept in the greenhouse for 2 months until they were
established properly. The shoots were defoliated and allowed to grow for 2 weeks. The first drought experiment was conducted in a manner similar to that described in Example 3. The seedlings did not receive water for a period of up to 3 weeks at which time the plant and soil were dried. Several times after water retention, a normal watering schedule was resumed. One week after resuming irrigation, the lengths of the limbs, and the fresh and dry weights of the shoots were determined. In an equivalent degree of drought aggression, tolerant plants were able to resume normal growth while susceptible plants had died or suffered noticeable damage resulting in shorter leaves and less dry matter. The proline content of the leaves and the opening of the stoma were also measured several times during the drought aggression. The tolerant plants maintained a lower proline content and a larger stomatal opening than the non-transgenic control plants. A second experiment that imposes aggression by drought in the transgenic plants was by treatment with a PEG solution as described in the previous examples. Salinity and cold tolerance were measured using methods as described in example 3. Plants that had tolerance to salinity or cold had yields
of higher seeds, photosynthesis and dry matter production than susceptible plants. Example 6 Design of stress-tolerant soybean plants by over-expressing genes related to aggression from Saccharomyces cerevisiae or E. coli. The soybean was transformed according to the following modification of the method described in the Texas A &M US patent 5,164,310. Various varieties of commercial soybeans are sensitive to transformation by this method. The Jack variety (available from the Illinois Seed Foundation) is one commonly used for transformation. The seeds were sterilized by immersion in 70% (v / v) of ethanol for 6 minutes and in 25% of the commercial decolorizing agent (NaOCl) supplemented with 0.1% (v / v) of Tween for 20 minutes, followed by rinsing 4 times with sterile double distilled water. Seedlings were propagated for seven days by removing the radicle, hypocotyledon and cotyledon for each seedling. Then, the epicotyledon with a cotyledon was transferred into a fresh germination medium in petri dishes and incubated at 25 ° C under a 16 hour photoperiod (approximately 100 μ? -? - 23-1) for three weeks. The axillary protuberances (approximately 4 mm long) were cut from 3-4 week old plants. The axillary protuberances were excised and incubated in Agrobacterium BL4404 culture.
Many different binary vector systems have been described for plant transformation (eg, An, G, in Agrobacterium Protocols, Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, New Sweater) . Many are based on the pBIN19 vector described by Bevan (Nucleic Acid Research, 1984. 12: 8711-8721) which includes a cassette of plant gene expression flanked by left and right margin sequences from the Ti plasmid of Agrobacterium tumefaciens. A cassette of gene expression of the plant consists of at least two genes - a selection marker gene and a plant promoter that regulates the transcription of the cDNA or genomic DNA of the characteristic gene. Several selection marker genes can be used including the Arabidopsis gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (U.S. Patent Nos. 57673666 and 6225105). Similarly, several promoters can be used to regulate the characteristic gene to provide constitutive, tissue-building or environmental regulation of genetic transcription. In this example, the 34S promoter (GenBank accession numbers M59930 and X16673) was used to provide constitutive expression of the characteristic gene. After the co-culture treatment, the explants were washed and transferred in a medium of
selection supplemented with 500 mg / 1 of timentina. The shoots were removed and placed in a shoot extension medium. The longer shoots of 1 cm were placed in a rooting medium for two to four weeks before transplanting to the ground. The primary transgenic plants (TO) were analyzed by PCR to confirm the presence of T-DNA. These results were confirmed by Southern hybridization in which the DNA is electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The DIG Probé Synthesis PCR (Roche Diagnostics) is used to prepare a probe labeled with digoxigenin by PCR, and is used as recommended by the manufacturer. Tolerant plants had higher seed yields, maintained their stoma opening and showed only slight changes in osmotic potency and proline levels, while susceptible non-transgenic control plants closed their stoma and exhibited increased osmotic potential and proline levels. Tolerance to drought, salinity and cold were measured using methods as described in Example 3. Plants that had tolerance to salinity or cold had higher seed yields, photosynthesis and dry matter production than susceptible plants.
Example 7 Design of stress-tolerant Colza / Canola Seed plants by overexpressing genes related to aggression from Saccharomyces cerevisiae or E. coli. The cotyledonous and hypocotyledon petioles of young seedlings of 5-6 days were used as explant for tissue culture and were transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The variety to be commercial (Agriculture Canada) is the standard variety used for transformation but other varieties can be used. Agrobacterium tumefaciens LBA4404 containing a binary vector was used for transformation of canola. Many different binary vector systems have been described for plant transformation (eg, An, G, in Agrobacterium Protocols, Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, New Sweater) . Many are based on the vector pBIN19 described by Bevan (Nucleic Acid Research, 1984, 12: 8711-8721) which includes a cassette of gene expression of the plant flanked by the left and right margin sequences from the Ti plasmid of Agrobacterium tumefaciens . A gene expression cassette consists of at least two genes - a selection marker gene and a plant promoter that regulates the transcription of the genomic cDNA or genomic DNA
characteristic. Several selection marker genes can be used including the Arabidopsis gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (U.S. Patent Nos. 57673666 and 6225105). Similarly, several promoters can be used to regulate the characteristic gene to provide constitutive, developmental, tissue or environmental regulation of genetic transcription. In this example, the 34S promoter (GenBank accession numbers M59930 and X16673) was used to provide the constitutive expression of the characteristic gene. The canola seeds were superficially sterilized in 70% ethanol for 2 minutes, and then in 30% Clorox with a drop of Tween-20 for 10 minutes, followed by three rinses with sterilized distilled water. The seeds were then germinated in vitro for 5 days in medium medium MS medium without hormones, 1% sucrose, 0.7% Phytagar at 23 ° C, 16 hours light. The cotyledon petiole explant with the cotyledon bound was excised from in vitro plantlets, and inoculated with Agrobacterium by dipping the cut end of the petiole explant into the bacterial suspension. The explants were then cultured for 2 days in an MSBAP-3 medium containing 3 mg / 1 BAP, 3% sucrose, 0.7% Phytagar at 23 ° C, 16 hours light. After two days of co-culture with Agrobacterium, the petiole explants were transferred to a
MSBAP-3 medium containing 3 mg / 1 BAP, cefotaxime, carbenicillin, or timentin (300 mg / 1) for 7 days, and then cultured in an MSBAP-3 medium with cefotaxime, carbenicillin or timentin and the selection agent until the regeneration of buds. When the shoots were 5-10 mm long, they were cut and transferred in an outbreak elongation medium (MSBAP-0.5, containing 0.5 mg / 1 BAP). The shoots of approximately 2 cm in length were transferred to the rooting medium (MSO) for root induction. Samples of the primary transgenic plants (T0) were analyzed by PCR to confirm the presence of T-DNA. These results were confirmed by Southern hybridization in which the DNA was electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The DID Probé Synthesis PCR (Roche Diagnostics) is used to prepare a probe labeled with digoxigenin by PCR, and is used as recommended by the manufacturer. The transgenic plants were then evaluated for their improved stress tolerance according to the method described in Example 3. The tolerant plants had higher seed yields, maintained their stoma opening and showed only slight changes in osmotic potential and proline levels, while the
non-transgenic susceptible control plants closed their stoma and exhibited increased osmotic potential and proline levels. Tolerance to drought, salinity and cold were measured using methods as described in example 3 above. Plants that had tolerance to salinity or cold had higher yields of seeds, photosynthesis and production of dry matter than susceptible plants. EXAMPLE 8 Design of stress-tolerant corn plants by over-expressing genes related to aggression from Saccharomyces cerevisiae or E. coli The transformation of corn (Zea Mays L.) was carried out with a modification of the method described by Ishida et al. (1996. Nature Biotech 14745-50). The transformation is genotype-dependent in corn and only specific genotypes are sensitive to transformation and regeneration. The congenital line A188 (University of Minnesota) or hybrids with A188 as a percentage 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. The ears were harvested from maize plants in about 11 days after pollination (DAP) when the length of immature embryos
it is approximately 1 to 1.2 mm. The immature embryos are co-cultivated with Agrobacterium tumefaciens that carry "super binary" vectors and transgenic plants are coated by organogenesis. The Japan Tobacco super binary vector system is described in WO WO94 / 00977 and WO95 / 06722. The vectors were constructed as described. Several selection marker genes can be used including the maize gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (US Patent 6025541). Similarly, several promoters can be used to regulate the characteristic gene to provide constitutive, developmental, tissue or environmental regulation of genetic transcription. In this example, the 34S promoter (Access GenBank numbers M59930 and X16673) was used to provide constitutive expression of the characteristic gene. Embryos extirpated in a callus induction medium, then a corn regeneration medium, are cultivated, which contain imidazolinone as a selection agent. The petri dishes are incubated in the light at 25 ° C for 2-3 weeks, or until the development of shoots. The green shoots are transferred from each embryo in a medium of rooting corn and incubated at 25 ° C for 2-3 weeks, until buds develop. The rooted shoots are transplanted to the earth in the greenhouse. IT seeds are produced from plants that exhibit tolerance to
Imidazolinone herbicides and which are PCR positive for the transgene. The transgenic TI plants were then evaluated for their improved tolerance to stress according to the method described in Example 3. The TI generation of single locus insertions of the T-DNA will segregate for the transgene in a 3: 1 ratio. Those progenies that contain one or more copies of the transgene are tolerant to the herbicide imidazolinone, and exhibit greater tolerance of aggression to drought than those progenies that lack transgenes. Tolerant plants had higher seed yields, maintained their stoma opening and showed only slight changes in osmotic potential and proline levels, while susceptible non-transgenic control plants closed their stoma and exhibited increased osmotic and proline levels. T2 homozygous plants exhibited similar phenotypes. Hybrid plants (progeny Fl) of homozygous transgenic plants and non-transgenic plants also exhibit tolerance to increased environmental aggression. Salinity and cold tolerance were measured using methods as described in example 3 above. Plants that had tolerance to drought, salinity or cold had higher yields of seeds, photosynthesis and production of dry matter than plants
susceptible. Example 9 Design of stress-tolerant wheat plants by over-expressing genes related to aggression from Saccharomyces cerevisiae or E. coli Wheat transformation is performed with the method described by Ishida et al. (1996 Nature Biotech, 14745-50). The Bobwhite variety (available from CYMMIT, Mexico) is commonly used in processing. The immature embryos are co-cultivated with Agrobacterium tumefaciens that carry "super binary" vectors, and transgenic plants are recovered by organogenesis. The super binary vector system of Tobacco from Japan is described in patents WO WO94 / 00977 and WO95 / 06722. The vectors were constructed as described. Several selectable marker genes can be used including the maize gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (North American patent 6025541). Similarly, several promoters can be used to regulate the characteristic gene to provide constitutive, developmental, tissue or environmental regulation of genetic transcription. In this example, the 34S promoter (Access GenBank numbers M59930 and X16673) was used to provide constitutive expression of the characteristic gene. After incubation with Agrobacterium, the
Embryos are grown in a callus induction medium, then the regeneration medium, which contains imidazolinone as a selection agent. Petri dishes are incubated in light at 25 ° C for 2-3 weeks, or until buds develop. The green shoots are transferred from each embryo to the rooting medium and incubated at 25 ° C for 2-3 weeks, until the shoots develop. The rooted shoots are transplanted to the earth in the greenhouse. IT seeds are produced from plants that exhibit tolerance to imidazolinone herbicides and which are PCR positive for transgenes. The transgenic IT plants were then evaluated for their improved tolerance to stress according to the method described in example 3 above. The TI generation of single locus insertions of the T-DNA will segregate for the transgene in a 3: 1 ratio. Those progenies that contain one or two copies of the transgene are tolerant of the herbicide imidazolinone, and exhibit greater tolerance of aggression by drought than those progenies that lack transgenes. Tolerant plants had higher seed yields, maintained their stoma opening and showed only slight changes in osmotic potential and proline levels, while susceptible non-transgenic control plants closed their stoma and exhibited increased osmotic potential and proline levels. The
T2 homozygous plants exhibited similar phenotypes. The tolerance of salinity and cold were measured using methods as described in the previous examples. Plants that had tolerance to drought, salinity or cold had higher yields of seeds, photosynthesis and production of dry matter than susceptible plants. Example 10 Identification of Identical and Heterologous Genes Genetic sequences can be used to identify identical or heterologous genes from cDNA or genomic libraries. Identical genes (e.g., full length cDNA clones) can be isolated through nucleic acid hybridization using e.g. cDNA libraries. Depending on the abundance of the gene of interest, 100,000 up to 1,000,000 recombinant bacteriophages are formed into plates and transferred to nylon membranes. After denaturing with alkali, the DNA is immobilized on the membrane, for example by cross-linking DNA. Hybridization is carried out at conditions of high severity. In aqueous solution, the hybridization and washing is carried out in an ionic strength of 1 M NaCl and a temperature of 68 ° C. Hybridization probes are generated, for example, by transcriptional labeling by radioactive cuts (32P) (High Prime, Roche, Mannheim, Germany). Signs are detected by autoradiography.
The partially identical or heterologous genes that are related but not identical can be identified in a manner analogous to the procedure described above using low severity hybridization and washing conditions. For aqueous hybridization, the ionic strength is normally maintained at 1 of NaCl while the temperature is progressively decreased from 68 to 42 ° C. The isolation of genetic sequences with homology (or identity / sequence similarity) only in a domain other than (eg, 10-20 amino acids) can be carried out using radiosynthetic labeled oligonucleotide probes. Radiolabeled oligonucleotides are prepared by phosphorylating the 5-prime end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides are annealed and ligated to form concatemers. The double-stranded concatemers are then radiolabelled, for example, by transcription by cuts. Hybridization is normally performed at conditions of low severity using high oligonucleotide concentrations. Oligonucleotide hybridization solution: 6 x 0.01 M SSC 1 mM EDTA sodium phosphate (pH 8) 0.5% SDS
100 pg / ml denatured salmon sperm DNA 0.1% fat-free dry milk During hybridization the temperature is decreased in stages at 5-10 ° C below the estimated Tm oligonucleotide or below the ambient temperature followed by steps of washing and auto-radiography. Washing is performed with low severity such as 3 washing steps using 4 x SSC. Additional details are described 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 iley & Sons. Example 11 Identification of Identical Genes by Expression Libraries of Selection with Antibodies c-DNA clones can be used to produce the recombinant polypeptide for example, in E. coli (for example, Qiagen QIAexpress pQE system). The recombinant polypeptides are then affinity purified normally with Ni-NTA affinity chromatography (Qiagen). The recombinant polypeptides are then used to produce specific antibodies for example, using standard techniques for rabbit immunization. The antibodies are purified by affinity using a Ni-NTA column saturated with the recombinant antigen as described by Gu
et al., 1994, BioTechniques 17: 257-262. The antibody can then be used to select expression cDNA libraries to identify identical or heterologous genes through an immunological selection (Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, FM et al., 1994, "Current Protocols in Molecular Biology," John Wiley &; Sons). Example 12 In vivo mutagenesis In vivo mutagenesis in microorganisms can be performed by passage of the plasmid DNA (or other vector) through E. coli or other microorganisms (e.g., Bacillus spp., Or yeasts such as Saccharomyces cerevisiae) which they deteriorate in their abilities to maintain the integrity of their genetic information. Strains of typical mutants have mutations in the genes for the DNA repair system (eg, mutHLS, mutD, mutT, etc., for reference, see Rupp, WD, 1996, DNA repair mechanisms, in: Escherichia coli and Salmonella, p 2277-2294, ASM: Washington). Such strains are well known to those skilled in the art. The use of such strains is illustrated, for example, in Greener, A and Callahan, M., 1994, Strategies 7: 32-34. The transfer of mutated DNA molecules in plants is preferably done after selection and experimentation in microorganisms. The
Transgenic plants are generated according to several examples within the implementation of this document. Example 13 Design of stress-tolerant Arabidopsis plants by over-expressing genes encoding aggression-related proteins from Brassica napus, Glycine max, Zea mays or Oryza sativa using stress-inducible and tissue-specific promoters. Transgenic Arabidopsis plants that express genes encoding aggression-related proteins from Brassica napus, Glycine max, Zea mays and Oryza sativa are created as described in Example 1 to express the transgenes encoding proteins related to low stress the control of a tissue-specific or stress-inducible promoter. The constitutive expression of a transgene can cause harmful side effects, which can be avoided by the use of these promoters. Stress inducible expression is achieved using promoters selected from those listed above in Table 1. T2 generation plants are produced and treated with drought aggression in two experiments. For the first drought experiment, plants are deprived of water until the plant and soil dry out. Several times after water retention, a normal watering schedule will resume
and the plants are grown until ripe. The yield of seeds is determined as seeds per plant. In an equivalent degree of drought aggression, tolerant plants are able to resume normal growth and produce more seeds than non-transgenic control plants. The proline content of the leaves and the stoma opening is also measured several times during the drought aggression. Tolerant plants maintain a lower proline content and a larger stoma opening than non-transgenic control plants. An alternative method for imposing aqueous aggression on transgenic plants by treatment with water containing osmolyte such as polyethylene glycol (PEG) at the specific aqueous potential. Since PEG can be toxic, plants are only given a short-term exposure and then normal watering is resumed. As in the above, the seed yields are measured from mature plants. The response is measured again during the stress period by typical measurements, such as by stoma opening or osmotic potential, or biochemical measurements, such as proline accumulation. Tolerant plants have higher seed yields, maintain their stoma opening and show only slight changes in osmotic potential and proline levels, while susceptible non-transgenic control plants close
their stoma and exhibit osmotic potential and increased proline levels. Transgenic plants with a constitutive promoter that controls transcription of the transgene are compared to those plants with a drought inducible promoter in the absence of stress. The results indicate that metabolite changes and gene expression do not occur when plants with the stress inducible promoter were cultured in the absence of stress. These plants also have higher seed yields than those with the constitutive promoter. Example 14 Over-expression of genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa provides tolerance of multiple abiotic aggressions. Plants that exhibit tolerance of abiotic aggression often exhibit tolerance of another environmental aggression or a herbicide that generates oxygen free radicals. This phenomenon of cross-tolerance is not understood on a mechanistic level (McKersie and Leshem, 1994). However, it is reasonable to expect that plants exhibiting improved drought tolerance due to the expression of a transgene could also exhibit low temperature tolerance, freezing, salt, airborne contaminants such as
ozone and other abiotic aggressions. In support of this hypothesis, the expression of several genes is over- or un-regulated by multiple factors of abiotic aggression including cold, salt, osmosis, ABA, etc. (eg Hong et al. (1992) Developmental and organ-specific expression or fan ABA- and stress induced protein in barley Plant Mol Biol 18: 663-674; Jagendorf and Takabe (2001) Inducers of glycinebetaine synthesis in barley Plant Physiol 127: 1827-1835); Mizoguchi et al. (nineteen ninety six) . A gene encoding a mitogen-activated protein kinase is simultaneously induced with genes for a mitogen-activated protein kinase and a ribosomal protein kinase S6 by contact, cold and water aggression in Arabidopsis thaliana. Proc Nati Acad Sci U S A 93: 765-769; Zhu (2001) Cell signaling under salt, water and cold stresses, Curr Opin Plant Biol 4: 401-406). Transgenic Arabidopsis plants that over express genes encoding aggression-related proteins from Brassica napus, Glycine max, Zea mays and Oryza sativa are created as described in example 1 and tested for tolerance to aggression by salt and cold . To determine the salt tolerance, Arabidopsis thaliana seeds are sterilized (incubated in 100% decolorizing agent, 0.1% TritonXlOO for five minutes (twice) and rinsed five times with ddH20). Seeds are plated in a non-selective medium (1/2 MS,
0. 6% of phytagar, 0.5 g / 1 of MES, 1% of acarose, 2 g / ml of benamilo). The seeds are allowed to germinate for approximately ten days. In the 4-5 stage of the leaf, the transgenic plants are sown in pots of 5.5 cm in diameter and allowed to grow (22 ° C, continuous light) for approximately seven days, watering when necessary. To begin the test, two liters of 100 mM NaCl and 1/8 deMS are added to the tray under the pots. To the tray containing the control plants, three liters of 1/8 MS is added. The concentrations of NaCl supplement are increased in steps by 50 mM every 4 days up to 200 mM. After saline treatment with 200 mM, the fresh and dry weights of the plants as well as seed yields are determined. Transgenic plants that overexpress genes encoding stress-related proteins from Brassica napus, Glycine max, Zea mays and Oryza sativa show higher fresh and dry weights and more seed yield compared to wild type or false transformed plants . To determine tolerance to cold, seeds of transgenic and cold lines are germinated and cultivated for about 10 days to stage 4-5 of the leaf as in the above. The plants are then transferred to cool temperatures (5 ° C) and are cultivated through the stages of flowering and assent of seeds of
development. Photosynthesis is measured using chlorophyll fluorescence as an indicator of photosynthetic capacity and integrity of photosystems. The yield of seeds and the dry weight of the plant are measured as an indicator of the biomass production of the plant. It is found that over-expression of genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa facilitate tolerance to salt and cold as well as drought. Plants that have tolerance to salinity or cold have also higher seed yields, photosynthesis and dry matter production than susceptible plants. Example 15 Design of stress-tolerant alfalfa plants by over-expressing genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa A clone of regenerating alfalfa (Medicago sativa) is transformed using McKersie et al. al., 1999 (Plant Physiol 119: 839-847). The regeneration and transformation of alfalfa is the dependent genotype and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the Rangelander variety (Agriculture Canada) or any other variety of commercial alfalfa as described by Brown and 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 Ann J Bot 65: 654-659). Petiole explant was co-cultured 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 (eg, n, G. in Agrobacterium Protocols, Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, New Sweater) . Many are based on the vector pBIN19 described by Bevan (Nucleic Acid Research, 1984. 12: 8711-8721) which includes a cassette of gene expression of the plant flanked by the left and right margin sequences from the Ti plasmid of Agrobacterium tumefaciens . A cassette of gene expression of the plant consists of at least two genes - a selection marker gene and a plant promoter that regulates the transcription of the cDNA or genomic DNA of the characteristic gene. Several selection marker genes can be used, including the Arabidopsis gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (U.S. Patent Nos. 57673666 and 6225105). Similarly, several promoters can be used to regulate the gene
characteristic that provides constitutive, developmental, tissue or environmental regulation of genetic transcription. In this example, the 34S promoter (accession number GenBank 59930 and X16673) was used to provide constitutive expression of the characteristic gene. The explants were co-cultured for 3 days in the dark in an SH induction medium containing 288 mg / 1 of Pro, 53 mg / L of thioproline, 4.35 g / 1 of K2S04, and 100 μp? of acetosinginone. The explants were washed in medium-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyrininone, but with an appropriate selection agent and an appropriate antibiotic to inhibit growth. of Agrobacterium. After several weeks, the somatic embryos are transferred in a development medium BOÍ2Y that do not contain growth regulators, nor antibiotics, and 50 g / 1 of sucrose. The somatic embryos were subsequently germinated in medium-strength Murashige-Skoog medium. Rooted seedlings were transplanted in pots and grown in a greenhouse. T0 transgenic plants were propagated by protrusion clippings and rooted in a Turface growth medium. The plants were defoliated and cultured at a height of approximately 10 cm (approximately 2 weeks after defoliating). The plants were then subjected to
drought aggression in two experiments. For the first drought experiment, the seedlings received no water for a period of up to 3 weeks at which time the plant and soil were dried. Several times after water retention, a normal watering schedule was resumed. One week after resuming the risk, the fresh and dry weights of the outbreaks were determined. In an equivalent degree of drought aggression, tolerant plants were able to resume normal growth while susceptible plants had died or suffered significant damage resulting in less dry matter. The proline content of the leaves and the opening of the stoma were also measured several times during the drought aggression. The tolerant plants maintained a lower proline content and a larger stoma opening than the non-transgenic control plants. An alternative method to impose aqueous aggression on transgenic plants is by treatment with a solution in specific aqueous potential, which contains an osmolyte such as polyethylene glycol (PEG). PEG treatment was given to any detached leaves (eg, Djilianov et al., 1997 Plant Science 129: 147-156) or roots (Wakabayashi et al., 1997 Plant Physiol 113: 967-973). Since PEG can be toxic, plants are only given a short-term exposure. The response was measured
as physical measurements such as stoma opening or osmotic potential, or biochemical measurements such as proline accumulation. Tolerant plants maintained their stoma opening and showed only slight changes in osmotic potential, while non-transgenic susceptible control plants closed their stoma and exhibited increased osmotic potential. In addition, changes in proline and other metabolites are lower in tolerant transgenic plants than in non-transgenic control plants. The tolerance of salinity and cold are measured using methods as described in example 3. It is found that alfalfa plants that overexpress genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa are more resistant to aggression by salinity and cold that non-transgenic control plants. Plants that have tolerance to salinity or cold have also higher seed yields, photosynthesis and dry matter production than susceptible plants. Example 16 Design of stress tolerant weed plants by over-expressing genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa The seeds of different varieties of weeds can be used as explant sources for transformation,
including the commercial variety Gunne available from the seed company Svalof eibull of the variety Affinity. Seeds are surface sterilized sequentially with 1% Tween-20 for 1 minute, 100% decolorizing agent for 60 minutes, 3 rinses with 5 minutes each with deionized and distilled H20, and then germinated for 3-4 days in humidity, the sterile filter paper in the dark. The seedlings are further sterilized for 1 minute with 1% Tween-20, 5 minutes with 75% decolorizing agent, and rinsed 3 times with ddH20, 5 minutes each. Superficially sterilized seeds are placed in the callus induction medium containing basal salts and Murashige and Skoog vitamins, 20 g / l sucrose, 150 mg / l asparagine, 500 mg / l casein hydrolyzate, 3 g / l Phytagel, 10 mg / 1 BAP, and 5 mg / 1 dicamba. The plates are incubated in the dark at 25 ° C for 4 weeks for seed germination and induction of embryogenic callus. After 4 weeks in the callus induction medium, the shoots and roots of the seedlings were cut, the callus was transferred to fresh medium, kept in culture for another 4 weeks, and then transferred to MSO medium in the light for 2 hours. weeks Several pieces of callus (11-17 weeks) are screened through a 10 mesh screen and placed in a callus induction medium, or cultured in 100 ml.
my induction medium of liquid tartar callus (same medium as for callus induction with agar) in a 250 ml flask. The flask is wrapped in aluminum foil and stirred at 175 rpm in the dark at 23C for 1 week. The liquid culture is sifted with a 40 mesh screen that collects the cells. The fraction collected in the sieve is formed into plates and cultivated in a medium of induction of solid tares callus for 1 week in the dark at 25 ° C. The callus is then transferred to and grown in an MS medium containing 1% sucrose for 2 weeks. The transformation can be achieved with any Agrobacterium with particle bombardment methods. An expression vector is created containing a constitutive plant promoter and the gene cDNA in a pUC vector. Plasmid DNA was prepared from E. coli cells using the Qiagen kit according to the manufacturer's instruction. Approximately 2 g of the embryogenic cells are scattered in the center of a sterile filter paper in a Petri dish. An aliquot of liquid MSO with 10 g / 1 of sucrose is added to the filter paper. The gold particles (1.0 μp in size) are coated with plasmid DNA according to the method of Sanford et al., 1993 and are supplied to the embryogenic callus with the following parameters: 500 g of particles and 2 g of DNA per stem, 1300 psi and a target distance of 8.5 cm from the stopping silver to the
callus plaque and 1 stem per callus plate. After the bombardment, the calluses are transferred back to the fresh callus development medium and kept in the dark at room temperature for a period of 1 week. The callus is then transferred to growth conditions in the light at 25 ° C to initiate embryonic differentiation with the appropriate selection agent, for example, 250 nM of Arsenal, 5 mg / 1 of PPT or 50 mg / 1 of kanamycin. The shoots resistant to the selection agent sprouted and once they rotted they are transferred to the ground. 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 the DNA was electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The DIG Probé Synthesis PCR Kit (Roche Diagnostics) is used to prepare a probe labeled with digoxigenin by PCR, and is used as recommended by the manufacturer. Vegetative T0 weed plants are propagated vegetatively by removing stems. The transplanted stems are kept in the greenhouse for 2 months until they are properly established. The shoots were defoliated and allowed to grow for 2 weeks. The first drought experiment is conducted in a
similar to that described in Example 3. The seedlings do not receive water for a period of up to 3 weeks at which time the plant and the soil dry up. Several times after water retention, a normal watering schedule is resumed. One week after resuming irrigation, the lengths of the limbs, and the fresh and dry weights of the shoots are determined. In an equivalent degree of drought aggression, tolerant plants are able to resume normal growth while susceptible plants have died or suffered noticeable damage resulting in shorter leaves and less dry matter. The proline content of the leaves and the opening of the stoma are also measured several times during the aggression by drought. Tolerant plants maintain a lower proline content and a larger stomatal opening than non-transgenic control plants. A second experiment that imposes aggression by drought in the transgenic plants was by treatment with a PEG solution as described in the previous examples. The tolerance of salinity and cold were measured using methods as described in example 3. It is found that stress-related genes that over-express tares from Brassica napus, Glycine max, Zea mays or Oryza sativa are more resistant to aggression by salinity and cold control non-transgenic plants. The plants that have
tolerance to salinity or cold have higher seed yields, photosynthesis and dry matter production than susceptible plants. Example 17 Design of stress-tolerant soybean plants by over-expressing genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa Soybean is transformed according to the following modification of the method described in the Texas patent A &M US 5,164,310. Several varieties of commercial soybeans are sensitive to transformation by this method. The Jack variety (available from the Illinois Seed Foundation) is one commonly used for transformation. The seeds are sterilized by immersion in 70% (v / v) of ethanol for 6 minutes and in 25% of the commercial decolorizing agent (NaOCl) supplemented with 0.1% (v / v) of Tween for 20 minutes, followed by rinsing 4 times with sterile double distilled water. Seedlings were propagated for seven days by removing the radicle, hypocotyledon and cotyledon for each seedling. Then, the epicotyl with a cotyledon was transferred in a fresh germination medium in petri dishes and incubated at 25 ° C under a 16-hour photoperiod (approximately 100 uE-m-2s-l) for three weeks. The axillary protuberances (approximately 4 mm in length) are cut from 3-4 week old plants. The axillary protuberances are
extirpate and incubate in Agrobacterium culture BL4404. Many different binary vector systems have been described for plant transformation (eg, An, G, in Agrobacterium Protocols, Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, New Sweater) . Many are based on the pBIN19 vector described by Bevan (Nucleic Acid Research, 1984. 12: 8711-8721) which includes a cassette of plant gene expression flanked by left and right margin sequences from the Ti plasmid of Agrobacterium tumefaciens. A cassette of gene expression of the plant consists of at least two genes - a selection marker gene and a plant promoter that regulates the transcription of the cDNA or genomic DNA of the characteristic gene. Several selection marker genes can be used including the Arabidopsis gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (U.S. Patent Nos. 57673666 and 6225105). Similarly, several promoters can be used to regulate the characteristic gene to provide constitutive, tissue-building or environmental regulation of genetic transcription. In this example, the 34S promoter (GenBank accession numbers M59930 and X16673) was used to provide constitutive expression of the characteristic gene. After the co-culture treatment, the
The explants are washed and transferred in a selection medium supplemented with 500 mg / 1 of timentin. The shoots are removed and placed in a shoot extension medium. The longer shoots of 1 cm are placed in a rooting medium for two to four weeks before transplanting to the ground. The primary transgenic plants (TO) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization in which the DNA is electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The DIG Probé Synthesis PCR (Roche Diagnostics) is used to prepare a probe labeled with digoxigenin by PCR, and is used as recommended by the manufacturer. Stress-tolerant soybean plants that over-express genes related to aggression from Brassica napus, Glycine max, Zwa mays or Oryza sativa, have higher seed yields, maintain their stoma opening and show only light changes. in osmotic potential and proline levels, while non-transgenic susceptible control plants close their stoma and exhibit increased osmotic potential and proline levels. Tolerance to drought, salinity and cold are measured using methods as described in example 3.
Plants that have salinity tolerance or expanded cold have higher seed yields, photosynthesis, and dry matter production than susceptible plants. Example 18 Design of stress-tolerant rapeseed / Canola seed plants by over-expressing genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa. The cotyledonous and hypocotyled petioles of young seedlings of 5-6 days are used as explants for tissue culture and are transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial Westar variety (Agriculture Canada) is the standard variety used for transformation but other varieties can be used. Agrobacterium tumefaciens LBA4404 which contains a binary vector is used for transformation of canola. Many different binary vector systems have been described for plant transformation (eg, An, G, in AGrobacterium Protocols, Methods in Molecular Biology vol 44, pp 47-62, Gartland KMA and MR Davey eds. Humana Press, otowa, New Sweater) . Many are based on the vector pBIN19 described by Bevan (Nucleic Acid Research, 1984, 12: 8711-8721) which includes a cassette of gene expression of the plant flanked by
the left and right margin sequences from the Ti plasmid of Agrobacterium tumefaciens. A cassette of plant gene expression consists of at least two genes - a selection marker gene and a plant promoter that regulates the transcription of the cDNA or genomic DNA of the characteristic gene. Several selection marker genes can be used including the Arabidopsis gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (U.S. Patent Nos. 57673666 and 6225105). Similarly, several promoters can be used to regulate the characteristic gene to provide constitutive, developmental, tissue or environmental regulation of genetic transcription. In this example, the 34S promoter (GenBank accession numbers M59930 and X16673) is used to provide constitutive expression of the characteristic gene. The canola seeds are surface sterilized in 70% ethanol for 2 minutes, and then in 30% Clorox with a drop of Tween-20 for 10 minutes, followed by three rinses with sterilized distilled water. The seeds are then germinated in vitro for 5 days in medium medium MS medium without hormones, 1% sucrose, 0.7% Phytagar at 23 ° C, 16 hours light. The cotyledon petiole explant with the cotyledon are excised from in vitro plantlets, and inoculated with Agrobacterium submerging the cut end of the explant of the petiole in
the bacterial suspension. The explants are then cultured for 2 days in an MSBAP-3 medium containing 3 mg / 1 BAP, 3% sucrose, 0.7% Phytagar at 23 ° C, 16 hours light. After two days of co-culture with Agrobacterium, the petiole explant is transferred to MSBAP-3 medium containing 3 mg / 1 of BAP, cefotaxime, carbenicillin, or timentin (300 mg / 1) -for 7 days, and then they are grown in an MSBAP-3 medium with cefotaxime, carbenicillin or timentina and the selection agent until the regeneration of shoots. When the shoots are 5-10 mm in length, they are cut and transferred in an outbreak extension medium (MSBAP-0.5, which contains 0.5 mg / 1 BAP). The shoots of approximately 2 cm in length are transferred to the rooting medium (MSO) for root induction. Samples of 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 DID Probé Synthesis PCR (Roche Diagnostics) is used to prepare a probe labeled with digoxigenin by PCR, and is used as recommended by the manufacturer. The transgenic plants are then evaluated for their improved stress tolerance according to the method
described in Example 3. It is found that genes related to stress that over-expresses rapeseed / transgenic canola from Brassica napus, Glycine max, Zea mays or Oryza sativa are more tolerant to environmental aggression than control plants non-transgenic Tolerant plants have higher seed yields, maintain their stoma opening and show only slight changes in osmotic potential and proline levels, while susceptible non-transgenic control plants close their stoma and exhibit increased osmotic potential and proline levels. Plants that have tolerance to salinity or cold have higher seed yields, photosynthesis and dry matter production than susceptible plants. Example 19 Design of stress-tolerant corn plants by over-expressing genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa The transformation of corn (Zea Mays L.) is carried out with a modification of the method described by Ishida et al. (1996. Nature Biotech 14745-50). The transformation is genotype-dependent in corn and only specific genotypes are sensitive to transformation and regeneration. The congenital line A188 (University of Minnesota) or hybrids with A188 as a percentage are good sources of material
donor for transformation (Fromm et al., 1990 Biotech 8: 833-839), but other genotypes can be used successfully as well. The ears are harvested from maize plants in about 11 days after pollination (DAP) when the length of immature embryos is approximately 1 to 1.2 mm. The immature embryos are co-cultivated with Agrobacterium tumefaciens that carry "super binary" vectors and transgenic plants are coated by organogenesis. The Japan Tobacco super binary vector system is described in patents WO WO94 / 00977 and WO95 / 06722. The vectors are constructed as described. Several selection marker genes can be used including the maize gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (US Patent 6025541). Similarly, several promoters can be used to regulate the characteristic gene to provide constitutive, developmental, tissue or environmental regulation of genetic transcription. In this example, the 34S promoter (Access GenBank numbers M59930 and X16673) is used to provide constitutive expression of the characteristic gene. Embryos extirpated in a callus induction medium, then corn regeneration medium, which contain imidazolinone as a selection agent are cultured. The petri dishes are incubated in the light at 25 ° C for 2-3 weeks, or until the development of shoots. Sprouts
rooted are transplanted to the earth in the greenhouse. IT seeds are produced from plants that exhibit tolerance to imidazolinone herbicides and which are PCR positive for transgenes. The transgenic plants TI are then evaluated for their improved tolerance to stress according to the method described in Example 3. The TI generation of single locus insertions of the T-DNA will be segregated for the transgene in a 1: 2: 1 ratio. Those progenies that contain one or more copies of the transgene (3/4 of the progeny) are tolerant of the herbicide imidazolinone, and exhibit greater tolerance of aggression to drought than those progenies that lack transgenes. Tolerant plants have higher seed yields, maintain their stoma opening and show only slight changes in osmotic potential and proline levels, while susceptible non-transgenic control plants close their stoma and exhibit increased potential osmotic and proline levels. T2 homozygous plants exhibit similar phenotypes. Hybrid plants (progeny Fl) of homozygous transgenic plants and non-transgenic plants also exhibit tolerance to increased environmental aggression. The tolerance of salinity and cold are measured using methods as described in example 3 above. Again, transgenic corn plants that overexpress
genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa are found to be tolerant to environmental aggressions. Plants that have tolerance to drought, salinity or cold have higher seed yields, photosynthesis and dry matter production than susceptible plants. Example 20 Design of stress-tolerant wheat plants by over-expressing genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa Wheat transformation is carried out with the method described by Ishida et al. (1996 Nature Biotech, 14745-50). The Bobwhite variety (available from CYMMIT, Mexico) is commonly used in processing. The immature embryos are co-cultivated with Agrobacterium tumefaciens that carry "super binary" vectors, and transgenic plants are recovered by organogenesis. The super binary vector system of Tobacco from Japan is described in patents WO WO94 / 00977 and O95 / 06722. The vectors were constructed as described. Several selectable marker genes can be used including the maize gene encoding a mutated acetohydroxy synthase acid (AHAS) enzyme (North American patent 6025541). Similarly, several promoters can be used to regulate the characteristic gene to provide constitutive, developmental regulation of
tissue or environmental genetic transcription. In this example, the 34S promoter (Access GenBank numbers M59930 and X16673) was used to provide constitutive expression of the characteristic gene. After incubation with Agrobacterium, the embryos are cultured in a callus induction medium, then the regeneration medium, which contains imidazolinone as a selection agent. Petri dishes are incubated in light at 25 ° C for 2-3 weeks, or until buds develop. The green shoots are transferred from each embryo to the rooting medium and incubated at 25 ° C for 2-3 weeks, until the roots develop. The rooted shoots are transplanted to the earth in the greenhouse. IT seeds are produced from plants that exhibit tolerance to imidazolinone herbicides and which are PCR positive for transgenes. The transgenic plants are then evaluated for their improved tolerance to stress according to the method described in example 3 above. The TI generation of single locus insertions of the T-DNA will segregate for the transgene in a 3: 1 ratio. Those progenies that contain one or two copies of the transgene are tolerant of the herbicide imidazolinone, and exhibit greater tolerance of aggression by drought than those progenies that lack transgenes. The tolerant plants had more yields
elevated seeds, maintain their stoma opening and show only slight changes in osmotic potential and proline levels, while susceptible non-transgenic control plants close their stoma and exhibit increased osmotic potential and proline levels. The homozygous T2 plants exhibit similar phenotypes. The tolerance of salinity and cold are measured using methods as described in the previous examples. Plants that overexpress genes related to aggression from Brassica napus, Glycine max, Zea mays or Oryza sativa have tolerance to drought, salinity or cold and have higher seed yields displayed, photosynthesis and dry matter production than susceptible plants non-transgenic Example 21 Identification of Identical and Heterologous Genes Genetic sequences can be used to identify identical or heterologous genes from cDNA or genomic libraries. Identical genes (e.g., full length cDNA clones) can be isolated through nucleic acid hybridization using e.g. cDNA libraries. Depending on the abundance of the gene of interest, 100,000 up to 1,000,000 recombinant bacteriophages are formed into plates and transferred to nylon membranes. After denaturing with alkali, the DNA is immobilized on the membrane, for example by
DNA crosslinking. Hybridization is carried out at conditions of high severity. In aqueous solution, the hybridization and washing is carried out in an ionic strength of 1 M NaCl and a temperature of 68 ° C. Hybridization probes are generated, for example, by transcriptional labeling by radioactive cuts (32P) (High Prime, Roche, Mannheim, Germany). Signs are detected by autoradiography. Partially identical or heterologous genes that are similar but not identical can be identified in a manner analogous to the procedure described above using hybridization of low severity and washing conditions. For aqueous hybridization, the ionic strength is normally maintained at 1M NaCl while the temperature is progressively lowered from 68 to 42 ° C. The isolation of genetic sequences with homology (or identity / sequence similarity) only in a domain other than for example, 10-20 amino acids can be carried out using radiosynthetic labeled oligonucleotide probes. Radiolabeled oligonucleotides are prepared by phosphorylating the 5-prime end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides are annealed and ligated to form concatemers. The double-stranded concatemers are then radiolabelled, for example, by transcription by cuts. Hybridization is performed
usually at conditions of low severity using high oligonucleotide concentrations. Oligonucleotide hybridization solution: 6 x 0.01 M SSC of 1 m EDTA sodium phosphate (pH 8) 0.5% SDS 100 g / ml denatured salmon sperm DNA 0.1% dry fat-free milk During hybridization, the temperature is decreased in stages at 5-10 ° C below the estimated Tm oligonucleotide or below room temperature followed by washing steps and autoradiography. Washing is performed with low severity such as 3 washing steps using 4 x SSC. Additional details are described 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. Example 22 Identification of Identical Genes by Libraries of Expression of Selection with Antibodies c-DNA clones can be used to produce the recombinant polypeptide for example, in E. coli (for example, Qiagen QIAexpress pQE system). The polypeptides
The recombinants are then purified by affinity, usually with Ni-NTA affinity chromatography (Qiagen). The recombinant polypeptides are then used to produce specific antibodies for example, using standard techniques for rabbit immunization. The antibodies are affinity purified using a Ni-NTA column saturated with the recombinant antigen as described by Gu et al., 1994, BioTechniques 17: 257-262. The antibody can then be used to select expression cDNA libraries to identify identical or heterologous genes through an immunological selection (Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, FM et al., 1994, "Current Protocols in Molecular Biology," John Wiley &Sons). EXAMPLE 23 In vivo mutagenesis In vivo mutagenesis in microorganisms can be performed by passage of the plasmid DNA (or other vector) through E. coli or other microorganisms (eg, Bacillus spp., Or yeasts such as Saccharomyces cerevisiae) which they deteriorate in their abilities to maintain the integrity of their genetic information. Strains of typical mutants have mutations in the genes for the DNA repair system (eg, mutHLS, mutD, mutT, etc., for reference, see Rupp W.D., 1996,
DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington). Such strains are well known to those skilled in the art. The use of such strains is illustrated, for example, in Greener, A and Callahan, M., 1994, Strategies 7: 32-34. The transfer of mutated DNA molecules into plants is preferably done after selection and experimentation in microorganisms. Transgenic plants are generated according to several examples within the scope of this document.
Claims (28)
- CLAIMS 1. A transgenic plant cell wherein tolerance and / or resistance to environmental aggression is increased when compared to a corresponding untransformed wild type plant cell by transformation with a nucleic acid encoding Stress Related Protein (SRP) selected from the group consisting of: a) a nucleic acid molecule that encodes one of the polypeptides shown according to SEQ. FROM IDENT. DO NOT. 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214 and / or 218 and / or 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972 and / or 974 or a fragment thereof, which confers an increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild type plant cell in an organism a part of it; b) a nucleic acid molecule comprising one of the nucleic acid molecules shown according to SEC. FROM IDENT. DO NOT. Go 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217; and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973 c) a nucleic acid molecule whose sequence can be derived from a polypeptide sequence encoded by a nucleic acid molecule of (a) or (b) as a result of the degeneracy of the genetic code and confers an increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; d) a nucleic acid molecule which encodes a polypeptide which has at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and conferring an increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part of it; e) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under severe hybridization conditions and conferring increased tolerance and / or resistance to environmental aggression when compared to a plant cell type non-transformed wild type in an organism or a part thereof; f) a nucleic acid molecule which comprises a nucleic acid molecule which is obtained by amplifying nucleic acid molecules from a cDNA library or a genomic library using the primers as shown in table 2 and conferring a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; g) a nucleic acid molecule encoding a polypeptide which is isolated with the aid of monoclonal antibodies against a polypeptide encoded by one of the nucleic acid molecules of (a) to (f) and conferring increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; and h) a nucleic acid molecule which is obtainable by selecting a suitable nucleic acid library under severe hybridization conditions with a probe comprising one of the sequences of the nucleic acid molecule of (a) to (g) 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 (g) and conferring a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof. or comprising a sequence which is complementary to it.
- 2. The whole transgenic plant cell of claim 1, derived from a monocotyledonous plant.
- 3. The transgenic plant cell of any of claims 1 or 2 derived from a dicotyledonous plant. .
- The transgenic plant cell of any of claims 1-3, wherein the plant is selected from the group consisting of corn, wheat, rye, oats, triticale, rice, barley, soybeans, peanuts, cotton, rapeseed, cañola, cassava, pepper, sunflower , flax, borage, safflower, flaxseed, spring, rapeseed, wild rapeseed, calendula, solenáceas plants, potato, tobacco, eggplant, tomato, species Vicia, pea, alfalfa, coffee, cocoa, tea, Salix species, oil palm , coconut, perennial herbs, forage crops and Arabidopsis thaliana.
- 5. The transgenic plant cell of claim 1, derived from a gymnosperm plant.
- 6. A transgenic plant generated from a plant cell according to any of claims 1-4 and which is a monocytoid or dicotyledonous plant.
- 7. A transgenic plant of claim 6, which is selected from the group consisting of corn, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, cassava, pepper, sunflower , flax, borage, safflower, flaxseed, spring, rape seed, wild rapeseed, marigolds, solanaceous plants, potato, tobacco, eggplant, tomato, species Vicia, peas, alfalfa, coffee, cocoa, tea, Salix species, oil palm , coconut, perennial herbs, forage crops and Arabidopsis thaliana.
- 8. A transgenic plant generated from a plant cell according to any of claims 1-5 and which is a gymnosperm plant.
- 9. A seed produced by a transgenic plant of any of claims 5-8, wherein the seed is genetically homozygous for a transgene that confers tolerance and / or increased resistance to environmental aggression when compared to a wild type plant cell. non-transformed corresponding to an increased tolerance to environmental aggression when compared to a corresponding non-transformed wild-type plant.
- 10. An isolated nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule encoding one of the polypeptides according to SEQ. FROM IDENT. NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214 and / or 218 and / or 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 739, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972 and / or 974 or a fragment thereof, which confers an increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; b) a nucleic acid molecule comprising one of the nucleic acid molecules according to SEC. FROM IDENT. NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 45, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973; c) a nucleic acid molecule whose sequence can be derived from a polypeptide sequence encoded by a nucleic acid molecule of (a) or (b) as a result of the degeneracy of the genetic code and confers a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; d) a nucleic acid molecule which encodes a polypeptide which has at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and conferring a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; e) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under severe hybridization conditions and conferring increased tolerance and / or resistance to environmental aggression when compared to a plant cell type non-transformed wild type in an organism or a part thereof; f) a nucleic acid molecule which comprises a nucleic acid molecule which is obtained by amplifying nucleic acid molecules from a cDNA library or a genomic library using the primers as shown in table 2 and conferring a tolerance and / or increased resistance to aggression environmental when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; g) a nucleic acid molecule encoding a polypeptide which is isolated with the aid of monoclonal antibodies against a polypeptide encoded by one of the nucleic acid molecules of (a) to (f) and conferring increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; and h) a nucleic acid molecule which is obtainable by selecting a suitable nucleic acid library under severe hybridization conditions with a probe comprising one of the sequences of the nucleic acid molecule of (a) to (g) 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 (g) and conferring a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof. whereby the nucleic acid molecule is distinguished on the sequence according to SEC. FROM IDENT. DO NOT. 1, 5, 9, 13, 17, 21, 25,, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973 by one or more nucleotides,
- 11. An isolated nucleic acid molecule comprising a molecule of nucleic acid selected from the group consisting of: a) a nucleic acid molecule that encodes one of the polypeptides shown in SEQ. FROM IDENT. NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214 and / or 218 and / or 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972 and / or 974 or a fragment thereof, which confers a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; b) a nucleic acid molecule comprising one of the nucleic acid molecules according to SEC. FROM IDENT. NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973; c) a nucleic acid molecule whose sequence can be derived from a polypeptide sequence encoded by a nucleic acid molecule of (a) or (b) as a result of the degeneracy of the genetic code and confers an increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part of it; d) a nucleic acid molecule which encodes a polypeptide which has at least 50% identity with the amino acid sequence of the polypeptide encoded by the nucleic acid molecule of (a) to (c) and conferring a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; e) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (a) to (c) under severe hybridization conditions and conferring increased tolerance and / or resistance to environmental aggression when compared to a plant cell type non-transformed wild type in an organism or a part thereof; f) a nucleic acid molecule which comprises a nucleic acid molecule which is obtained by amplifying nucleic acid molecules from a cDNA library or a genomic library using the primers as shown in Table 2 and conferring increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; g) a nucleic acid molecule encoding a polypeptide which is isolated with the aid of monoclonal antibodies against a polypeptide encoded by one of the nucleic acid molecules of (a) to (f) and conferring increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof; and h) a nucleic acid molecule which is obtainable by selecting a suitable nucleic acid library under severe hybridization conditions with a probe comprising one of the sequences of the nucleic acid molecule of (a) to (g) 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 (g) and conferring a tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell in an organism or a part thereof.
- 12. A nucleic acid construct which confers the expression of the nucleic acid molecule of claim 10 or 11, comprising one or more regulatory elements, whereby the expression of the nucleic acid encoding SRP in a host cell results in tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell.
- 13. A vector comprising the nucleic acid molecule as claimed in claim 10 or 11 or the nucleic acid construct of claim 12, whereby the expression of the nucleic acid encoding SRP in a host cell results in tolerance and / or increased resistance to environmental aggression when compared to a corresponding non-transformed wild-type plant cell.
- 14. A host cell, which has been stably or temporarily transformed with the vector as claimed in claim 13 or the nucleic acid molecule as claimed in claim 10 or 11 or the nucleic acid construct of claim 12.
- 15. An isolated Stress Related Protein (SRP) which is selected from the group comprising SEC. FROM IDENT. NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214 and / or 218 and / or 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,360. , 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562., 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 740, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864, 866, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972 and / or 974 and / or homologs thereto.
- 16. An isolated Stress Related Protein (SRP) of claim 18, which is selected from yeast, preferably Saccharomyces cerevisiae or E. coli or Brassica napus, Glycime max, Zea mays or Oryza sativa.
- 17. A method for 'producing a transgenic plant with increased tolerance and / or resistance to environmental aggression compared to a corresponding non-transformed wild-type plant cell, where tolerance and / or resistance to environmental aggression is altered by expression of nucleic acid encoding the Stress Related Protein (SRP) and results in increased tolerance and / or resistance to environmental aggression when compared to a corresponding non-transformed wild type plant cell, comprising a) transforming a plant cell with an expression vector according to claim 16 and b) generating from the plant cell a transgenic plant with an increased tolerance to environmental aggression when compared to a corresponding non-transformed wild-type plant.
- 18. The method of claim 17, wherein the nucleic acid encoding the SRP is selected from the group comprising the nucleic acids according to SEQ. FROM IDENT. DO NOT. 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689 r 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713 r 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973 and / or homologs of the sequences mentioned above.
- 19. A method for modifying stress tolerance of a plant, which includes, modifying the level of expression of an SRP in the plant.
- The method of any of claims 17-19, wherein an expression vector is used according to any of claims 12 or 13.
- 21. The method of any of claims 17-20, wherein the tolerance to stress is decreased.
- 22. Use of a nucleic acid encoding SRP selected from the group comprising the nucleic acid of the SEC. FROM IDENT. NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217; and / of 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571 r 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595 r 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973 and / or homologs of the sequences mentioned above for preparing a plant cell with tolerance to increased environmental aggression.
- 23. Use of tolerance and / or increased resistance to environmental aggression and / or a nucleic acid encoding SRP selected from the group comprising the SEC nucleic acid. FROM IDENT. NO: 1, 5, 3, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265 r 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289 r 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599 r 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, VII, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973 homologues of the sequences mentioned above or parts thereof as markers for plant or plant cell selection with increased tolerance to environmental aggression.
- 24. Use of tolerance and / or increased resistance to environmental aggression and / or a nucleic acid encoding SRP selected from the group comprising the SEC nucleic acid. FROM IDENT. NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213 and / or 217 and / or 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267 (269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 847, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971 and / or 973 and / or homologs of the sequences mentioned above or parts thereof as markers for stress detection in plants or plant cells.
- 25. A nucleic acid construct which confers the expression of the nucleic acid molecule of claim 10 or 11, comprising one or more regulatory elements, whereby the expression of the nucleic acid encoding SRP in a host cell results in increased tolerance to environmental aggression when compared to a corresponding non-transformed wild-type host cell.
- 26. A vector comprising the nucleic acid molecule as claimed in claim 10 or 11 or the nucleic acid construct of claim 25, whereby expression of the nucleic acid encoding SRP in a host cell results in increased tolerance to environmental aggression when compared to a corresponding non-transformed wild-type host cell.
- 27. A plant cell comprising a nucleic acid construct of claim 25 or a vector of claim 26.
- 28. A plant, comprising a cell of claim 27.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US60/613.194 | 2004-09-24 | ||
US60/618.738 | 2004-10-14 |
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MX2007003358A true MX2007003358A (en) | 2008-10-03 |
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