DNA SEQUENCES AND PLASMIDS FOR THE PREPARATION OF PLANTS WITH CHANGED SUCROSE CONCENTRATION

FIELD OF THE INVENTION

[0001] The present invention relates to DNA sequences and plasmids, containing these DNA sequences, which by integration into a plant genome, cause the activity of the sucrose-phosphate-synthase (SPS) of the plant to be changed and thus affect the sugar metabolism of the plant. The invention further relates to transgenic plants, in which through introduction of the DNA sequences, changes in the activity of the sucrose-phosphate-synthase are produced.

[0002] Sucrose is of central importance for the plant and serves many functions. For the long distance transport of photoassimilates and/or energy between various organs in plants, sucrose is almost exclusively used. The sucrose, which is transported in a specific heterotrophic organ, determines the growth and the development of this organ. Thus it is known, e.g. from EP 442 592, that transgenic plants, in which the transport away of the sucrose from the exporting leaves is inhibited by expression of an apoplastic invertase, shows a strong reduction in the growth of e.g. roots or tubers in the case of potato plants. For tobacco plants, the principal importance of sucrose as the central function for the long distance transport of energy carriers within the plant is described (von Schaewen et al, 1990, EMBO J 9: 3033-3044).

[0003] Whilst it has been clearly shown that a reduction of the amount of sucrose imported in the heterotrophic organs, such as tubers and seeds, leads to loss of yield, it is not known whether an increase in the amount of sucrose in the photosynthetically active parts of the plant, mainly the leaves, leads to a better supply of heterotrophic organs and thus to an increase in yield.

[0004] A second central role for sucrose and/or the hexoses, glucose and fructose, derived from sucrose, is in the protection of plants against frost damage at low temperatures. Frost damage is one of the main limiting factors in agricultural productivity in the northern hemisphere. Temperatures below freezing lead to the formation of ice crystals. Since the growing ice crystals consist of pure water, water is abstracted from the cells as the temperature falls.

[0005] This dehydration has at least two potential damaging results:

[0006] a) all dissolved substances within a cell are strongly concentrated and the cell contracts following the loss of water. Highly concentrated salts and organic acids lead to membrane damage;

[0007] b) with rehydration from dew, the previously contacted cells reexpand. The cell membrane also expands again. The volume expansion puts a heavy mechanical load on the membrane.

[0008] It is thus clear that a freezing/dew cycle can lead to severe membrane damage of the cells and thus to damage to the plant.

[0009] It thus appears worth trying to hinder the freezing. One possible strategy is the increased formation of osmotically active substances in the cytosol of plant cells. This should lead to a lowering of the freezing point. Osmotically active substances include sucrose and/or the two hexoses derived from sucrose.

[0010] The increased formation of sucrose and/or the two hexoses at low temperatures is desirable in the growing plant. Another situation can exist in the harvested parts of a plant, especially in storage. For example, in potato tubers that are stored at 4-8° C., hexoses (glucose) accumulate. It would appear to be sensible, to see this as the answer to a lowering of the temperature (“cold-sweetening”).

[0011] The accumulation of sucrose and glucose has in the case of potato tubers economically undesirable results. Increased amounts of reducing sugars, such as glucose, in potatoes which are fried when preparing crisps, chips and the like, leads to an undesirable browning due to the Maillard reaction. Such products with a dark brown color are not generally acceptable to the consumer. Further the cooking strength is strongly dependent on the content of starch and/or its breakdown products which are important in determining the quality characteristics of the potato.

[0012] In relation to the economic aspects, sucrose thus possesses three especially important functions:

[0013] 1 as the transport form for the distant transport of photoassimilates,

[0014] 2 as an osmotically active substance with the desirable activity of lowering the freezing point in intact, growing plants, and

[0015] 3 in the undesirable formation of reducing sugars in stored harvested parts of a plant, e.g. the potato tubers, as a result of low temperatures.

[0016] The biosynthesis pathways for the formation of sucrose, either from the primary photosynthesis products (in the leaf) or by breakdown of starch (in the storage organs e.g. of potatoes), are known. An enzyme in sucrose metabolism is sucrose-phosphate-synthase (SPS). It forms sucrose-6-phosphate from UDP-glucose and fructose-6-phosphate, which in a second step sucrose. The isolation of SPS from maize and from MRNA from maize tissue is know application, processes for the puriified such as by centrifuging of homogena precipitation and chromatography are times enrichment of SPS from plant described by Salerno and Pontis (P]

[0017] In view of the significance of SPS for carbohydrate metabolism it is questionable whether plants tolerate a reduction in SPS activity in all or in certain organs. It is especially not known, whether it is possible to produce transgenic plants with a reduced SPS activity. Also the use of SPS for the modification of the functions of sucrose for lowering the freezing point in intact plants and for the formation of reducing sugars in harvested parts is not known.

[0018] For the preparation of plants with reduced SPS activity, i.e. plants with changed sucrose concentration, it is necessary to make available an SPS coding region of such plant species, for which processes are described, whereby transgenic plants can be grown in large numbers. In as much as a reduction of SPS activity can be achieved, by selection from a large amount, the possibility exists of obtaining plants with such a phenotype. Further organ specific promoters for gene expression should exist for the plant species, by which the possibility of an organ specific reduction of the SPS activity could be investigated.

[0019] A species which fulfils the stated requirements is Solanum tuberosum. The genetic modification of Solanum tuberosum by gene transfer using Aaobakteria is well described (Fraley et al., 1985, Crit Rev Plant Sci 4: 1-46). Promoters for leaf specific (Stockhaus et al., 1989, Plant Cell 1: 805-813), tuber specific (EP 375 092) and wound inducing (EP 375 091) gene expression are known.

[0020] The present invention now provides DNA sequences with which changes of SPS activity are actually and demonstrably possible and with which the sucrose concentration in the plant can be modified. It is concerned with sequences with the coding region of sucrose-phosphate-synthase (SPS) from Solanum tuberosum.

[0021] These DNA sequences can be introduced in plasmids and thereby combined with steering elements for expression in eukaryotic cells. Such steering elements are on the on one hand transcription promoters and on the other hand transcription terminators.

[0022] Each plasmid comprises:

[0023] a) a suitable promoter, that ensures that the coding sequence is read off at the suitable time point and/or in a specified development stage in the transgenic plants or in specified tissues of transgenic plants,

[0024] b) at least one coding sequence, that

[0025] i) is so coupled to the promoter that the formation of translatable RNA is allowed in a protein, whereby the protein demonstrates enzymatic activity, that leads to a modification of the sucrose concentration in the plant, or

[0026] ii) is so coupled to the promoter that the non-coding strand is read off, which leads to the formation of a so-called “anti-sense” RNA, which suppresses the formation of the coding protein of an endogenous gene in the plant, which is involved in the sucrose biosynthesis, and

[0027] c) a non-coding termination sequence, that contains the signals for the termination and polyadenylation of the transcript.

[0028] The present invention further provides plasmids in which there are the DNA sequences which change the SPS activity in the plant.

[0029] The coding sequences named under b) are the SPS sequences with the following nucleotide sequences:

1SPS 1 sequence (Seq. ID. No. 1):CTATTCTCTC CCCTCCTTTT TCTCCTCTCT TCAACCCCAA AACTTCCCTT TCAAAGCCTT60TGCTTTCCCT TTCTCACTTA CCCAGATCAA CTAAGCCAAT TTGCTGTAGC CTCAGAAAAC120AGCATTCCCA GATTGAAAAA GAATCTTTTT CAGTACCCAA AAGTTGGGTT TCTCATGTCC180AGCAAGGATT AGCTGCTCTA GCTATTTCTT TAGCCCTTAA TTTTTGTCCA GTTGTGTCTT240CTGATTCTGC ATTGGCATCT GAATTTGATG TGTTAAATGA AGGGCCACCA AAGGACTCAT300ATGTAGTTGA TGATGCTGGT GTGCTTAGCA GGGTGACAAA GTCTGATTTG AAGGCATTGT360TGTCTGATGT GGAGAAGAGA AAAGGCTTCC ACATTAATTT CATCACTGTC CGCAAGCTCA420CTAGCAAAGC TGATGCTTTT GAGTATGCTG ACCAAGTTTT GGAGAAGTGG TACCCTAGTG480TTGAACAAGG AAATGATAAG GGTATAGTTG TGCTTGTTAC AAGTCAAAAG GAAGGCGCAA540TAACCGGTGG CCCTGATTTT GTAAAGGCCG TTGGAGATAC TGTTCTTGAT GCTACCGTCT600CAGAGAACCT TCCAGTGTTG GCTACTGAAG AGAAGTACAA TGAAGCAGTT TTCAGCACTG660CCACACGTCT TGTTGCAGCC ATTGATGGCC TTCCTGATCC TGGTGGACCC CAACTCAAGG720ATAACAAAAG AGAGTCCAAC TTCAAATCCA GAGAGGAAAC TGATGAGAAA AGAGGACAAT780TCACACTTGT GGTTGGTGGG CTGTTAGTGA TTGCTTTTGT TGTTCCTATG GCTCAATACT840ATGCATATGT TTCAAAGAAG TGAACTGTCT GATTCTGGAA AGTTACATTT TCGTGAGATT900TGAGTAAGCA TGTATATTAT CGTGTACAAA ATGGTCCATT CGGAAATGAC TGATTC956ATG AGA TAT TTA AAA AGG ATA AAT ATG AAG ATT TGG ACC TCC CCT1001Met Arg Tyr Leu Lys Arg Ile Asn Met Lys Ile Trp Thr Ser Pro1 5 10 15AAC ATA ACG GAT ACT GCC ATT TCT TTT TCA GAG ATG CTG ACG CAA1046Asn Ile Thr Asp Thr Ala Ile Ser Phe Ser Glu Met Leu Thr Pro 16 25 30ATA AGT ACA GAC GGC TTG ATG ACT GAG ATG GGG GAG AGT AGT GGT1091Ile Ser Thr Asp Gly Leu Met Thr Glu Met Gly Glu Ser Ser Gly 35 40 45GCT TAT ATT ATT CGC ATT CCT TTT GGA CCA AGA GAG AAA TAT ATT1136Ala Tyr Ile Ile Arg Ile Pro Phe Gly Pro Arg Glu Lys Tyr Ile 50 55 60CCA AAA GAA CAG CTA TGG CCC TAT ATT CCC GAA TTT GTT GAT GGT1181Pro Lys Glu Gln Leu Trp Pro Tyr Ile Pro Glu Phe Val Asp Gly 65 70 75GCA CTT AAC CAT ATT ATT CAA ATG TCC AAA GTT CTT GGG GAG CAA1226Ala Leu Asn His Ile Ile Gln Met Ser Lys Val Leu Gly Glu Gln 80 85 90ATT GGT AGT GGC TAT CCT GTG TGG CCT GTT GCC ATA CAC GGA CAT1271Ile Gly Ser Gly Tyr Pro Val Trp Pro Val Ala Ile His Gly His 95 100 105TAT GCT GAT GCT GGC GAC TCA GCT GCT CTC CTG TCA GGT GCT TTA1316Tyr Ala Asp Ala Gly Asp Ser Ala Ala Leu Leu Ser Gly Ala Leu 110 115 120AAT GTA CCA ATG CTT TTC ACT GGT CAC TCA CTT GGT AGA GAT AAG1361Asn Val Pro Met Leu Phe Thr Gly His Ser Leu Gly Arg Asp Lys 125 130 135TTG GAG CAA CTG TTG CGA CAA GGT CGT TTG TCA AAG GAT GAA ATA1406Leu Glu Gln Leu Leu Arg Gln Gly Arg Leu Ser Lys Asp Glu Ile 140 145 150AAC TCA ACC TAC AAG ATA ATG CGG AGA ATA GAG GCT GAA GAA TTA1451Asn Ser Thr Tyr Lys Ile Met Ar gArg Ile Glu Ala Glu Glu Leu 155 160 165ACT CTT GAT GCT TCC GAA ATT GTC ATC ACT AGT ACA AGA CAG GAG1496Thr Leu Asp Ala Ser Glu Ile Val Ile Thr Ser Thr Arg Gln Glu 170 175 180ATT GAC GAG CAA TGG CGT TTG TAT GAT GGG TTT GAT CCA ATA TTA1541Ile Aso Glu Gln Trp Arg Leu Tyr Asp Gly Phe Asp Pro Ile Leu 185 190 195GAG CGT AAG TTA CGT GCA AGG ATC AAG CGC AAT GTC AGC TGT TAT1586Glu Arg Lys Leu Arg Ala Arg Ile Lys Arg Asn Val Ser Cys Tyr 200 205 210GGC AGG TTT ATG CCT CGT ATG GCT GTA ATT CCT CCT GGG ATG GAG1631Gly Arg Phe Met Pro Arg Met Ala Val Ile Pro Pro Gly Met Glu 215 220 225TCC CAC CAT ATT GTG CCA CAT GAA GGT GAC ATG GAT GGA GAA ACA1676Phe His His Ile Val Pro His Glu Gly Asp Met Asp Gly Glu Thr 230 235 240GAA GGA AGT GAA GAT GGG AAG ACC CCG GAT CCA CCT ATT TGG GCA1721Glu Gly Ser Glu Asp Gly Lys Thr Pro Asp Pro Pro Ile Trp Ala 245 250 255GAG ATT ATG CGC TTC TTT TCT ATT CCA AGG AAG CCT ATG ATA CTC1766Glu Ile Met Arg Phe Phe Ser Asn Pro Arg Lys Pro Met Ile Leu 260 265 270GCA CTT GCT AGG CCT GAT CCC AAG AAG AAC CTC ACT ACT TTA GTG1811Ala Leu Ala Arg Pro Asp Pro Lys Lys Asn Leu Thr Thr Leu Val 275 280 285AAA GCA TTT GGT GAA TGT CGT CCA TTG AGA GAG CTT GCT AAT CTT1856Lys Ala Phe Gly Glu Cys Arg Pro Leu Arg Glu Leu Ala Asn Leu 290 295 300ACT TTG ATA ATG GGT AAT CGA GAT AAT ATC GAC GAA ATG TCT AGC1901Thr Leu Ile Met Gly Asn Arg Asp Asn Ile Asp Glu Met Ser Ser 305 310 315ACC AAT TCT GCA CTT CTT CTT TCA ATC TTG AAA ATG ATA GAT AAG1946Thr Asn Ser Ala Leu Leu Leu Ser Ile Leu Lys Met Ile Asp Lys 320 325 330TAT GAT CTT TAT GGT CAA GTA GCT TAT CCT AAA CAC CAC AAG CAG1991Tyr Asp Leu Tyr Gly Gln Val Ala Tyr Pro Lys His His Lys Gln 335 340 345TCA GAT GTT CCT GAT ATC TAC CGT CTT GCT GCA AAG ACT AAG GGT2036Ser Asp Val Pro Asp Ile Tyr Arg Leu Ala Ala Lys Thr Lys Gly 350 355 360GTT TTT ATT AAT CCA GCT TTT ATT GAG CCT TTT GAA CTG ACT TTG2081Val Phe Ile Asn Pro Ala Phe Ile Glu Pro The Gly Leu Thr Leu 365 370 375ATT GAG GCA GCA GCT TAT GGT CTC CCA ATG GTA GCC ACA AAA ATT2126Ile Glu Ala Ala Ala Tyr Gly Leu Pro Met Val Ala Thr Lys Asn 380 385 390GGA GGA CCT GTT GAT ATA CAT AGG GTT CTT GAC AAT GGT CTC TTA2171Gly Gly Pro Val Asp Ile His Arg Val Leu Asp Asn Gly Leu Leu 395 400 405GTG GAT CCC CAT GAT CAG CAG GCA ATT GCT GAT GCT CTT TTG AAG2216Val Asp Pro His Asp Gln Gln Ala Ile Ala Asp Ala Leu Leu Lys 410 415 420TTG GTT GCT GAT AAG CAA CTG TGG GCT AAA TGC AGG GCA AAT GGA2261Leu Val Ala Asp Lys Gln Leu Trp Ala Lys Gys Arg Ala Asn Gly 425 430 435TTA AAA AAT ATC CAC CTT TTC TCA TGG CCC GAG CAC TGT AAA ACT2306Leu Lys Asn Ile His Leu Phe Ser Trp Pro Glu His Cys Lys Thr 440 445 450TAT CTA TCC CGG ATA GCT AGC TGC AAA CCA AGG CAA CCA CGC TGG2351Tyr Leu Ser Arg Ile Ala Ser Cys Lys Pro Arg Gln Pro Arg Trp 455 460 465CTG AGA TCC ATT GAT GAT GAT GAT GAA AAT TCA GAA ACA GAT TCA2396Leu Arg Ser Ile Asp Asp Asp Asp Glu Asn Ser Glu Thr Asp Ser 470 475 480CCT AGT GAT TCC TTG AGA GAT ATT CAT GAT ATA TCT CTG AAT TTG2441Pro Ser Asp Ser Leu Arg Asp Ile His Asp Ile Ser Leu Asn Leu 485 490 495AGA TTT TCA TTA GAT GGG GAA AAG AAT GAC AAT AAA GAA AAT GCT2486Arg Phe Ser Leu Asp Gly Glu Lys Asn Asp Asn Lys Glu Asn Ala 500 505 510GAT AAT ACA TTA GAC CCC GAA GTT CGA AGG AGC AAG TTA GAG AAT2531Asp Asn Thr Leu Asp Pro Glu Val Arg Arg Ser Lys Leu Glu Asn 515 520 525GCT GTT TTG TCC TTA TCT AAG GGT GCA CTG AAG AGC ACA TCA AAA2576Ala Val Leu Ser Leu Ser Lys Gly Ala Leu Lys Ser Thr Ser Lys 530 535 540TCT TGG TCG TCA GAC AAG GCA GAC CAA AAC CCT GGT GCT GGT AAA2621Ser Trp Ser Ser Asp Lys Ala Asp Gln Asn Pro Gly Ala Gly Lys 545 550 555TTC CCA GCG ATT AGG AGG AGG CGA CAT ATT TTT GTT ATT GCA GTG2666Phe Pro Ala Ile Arg Arg Arg Arg His Ile Phe Val Ile Ala Val 560 560 565GAT TGT GAT GCT AGC TCA GGA CTC TCT GGA AGT GTG AAA AAG ATA2711Asp Cys Asp Ala Ser Ser Gly Leu Ser Gly Ser Val Lys Lys Ile 570 575 530TTT GAG GCT GTA GAG AAG GAA AGG GCA GAG GGT TCC ATT GGA TTT2756Phe Glu Ala Val Glu Lys Glu Arg Ala Glu Gly Ser Ile Gly Phe 585 590 595ATC CTG GCT ACA TCT TTC AAT ATA TCA GAA GTA CAG TCT TTC CTG2801Ile Leu Ala Thr Ser Phe Asn Ile Ser Glu Val Gln Ser Phe Leu 600 605 610CTT TCA GAG GGC ATG AAT CCT ACT GAT TTT GAT GCT TAC ATA TGC2846Leu Ser Glu Gly Met Asn Pro Thr Asp Phe Asp Ala Tyr Ile Cys 615 620 625AAT AGT GGT GGT GAT CTT TAT TAT TCG TCC TTC CAT TCT GAG CAA2891Asn Ser Gly Gly Asp Leu Tyr Tyr Ser Ser Phe His Ser Glu Gln 630 635 640AAT CCT TTT GTA GTT GAC TTG TAC TAT CAC TCA CAT ATT GAG TAT2936Asn Pro Phe Val Val Asp Leu Tyr Tyr His Ser His Ile Glu Tyr 645 650 655CGT TGG GGG GGC GAA GGA TTG AGA AAG ACT TTG GTG CGT TGG GCC2981Arg Trp Gly Gly Glu Gly Leu Arg Lys Thr Leu Val Arg Trp Ala 660 665 670GCC TCT ATC ATT GAT AAG AAT GGT GAA AAT GGA GAT CAC ATT GTT3026Ala Ser Ile Ile Asp Lys Asn Gly Glu Asn Gly Asp His Ile Val 675 680 685GTT GAG GAT GAA GAC AAT TCA GCT GAC TAC TGC TAT ACT TTC AAA3071Val Glu Asp Glu Asp Asn Ser Ala Asp Tyr Cys Tyr Thr Phe Lys 690 695 700GTC TGC AAG CCT GGG ACG GTT CCT CCA TCT AAA GAG CTT AGA AAA3116Val Cys Lys Pro Gly Thr Val Pro Pro Ser Lys Glu Leu Arg Lys 705 710 715GTA ATG CGA ATT CAG GCA CTT CGT TGT CAC GCT GTT TAT TGT CAA3161Val Met Arg Ile Gln Ala Leu Arg Cys His Ala Val Tyr Cys Gln 720 725 730AAT GGG AGT AGG ATT AAT GTG ATC CCT GTA CTG GCA TCT CGG TCC3206Asn Gly Ser Arg Ile Asn Val Ile Pro Val Leu Ala Ser Arg Ser 735 740 745CAA GCA CTC AGG TAC TTA TAT CTG CGA TGG GGA ATG GAC TTG TCG3251Gln Ala Leu Arg Tyr Leu Tyr Leu Arg Trp Gly Met Asp Leu Ser 750 755 760AAG TTG GTG GTT TTC GTC GGA GAA AGT GGT GAT ACC GAT TAT GAA3296Lys Leu Val Val Phe Val Gly Glu Ser Gly Asp Thr Asp Tyr Glu 765 770 775GGA TTA ATC GGT GGT CTA CGC AAG GCT GTC ATA ATG AAA GGC CTC3341Gly Leu Ile Gly Gly Leu Arg Lys Ala Val Ile Met Lys Gly Leu 780 785 790TGC ACT AAT GCA AGC AGC TTA ATT CAC GGT AAT AGG AAT TAC CCG3386Cys Thr Asn Ala Ser Ser Leu Ile His Gly Asn Arg Asn Tyr Pro 795 800 805CTA TCT GAT GTT TTA CCA TTC GAC AGC CCT AAT GTC ATC CAA GCG3431Leu Ser Asp Val Leu Pro Phe Asp Ser Pro Asn Val Ile Gln Ala 810 815 820GAC GAG GAA TGT AGC AGC ACC GAA ATC CGT TGC TTA CTG GTG AAA3476Asp Glu Glu Cys Ser Ser Thr Glu Ile Arg Cys Leu Leu Val Lys 825 830 835CTA GCG GTA CTC AAA GGA TAATACCCTT CCCCCTTTGA TTGTCAAAAA3524Leu Ala Val Leu Lys Gly 840CCTATATGAG CTATAAGACT ATGCCATGAA AAGAATGGCC ATCCATTTGG CTTGTCTTTT3584GAAGCTGTTA ATACTTTTCA ACAGACTACA AAATGAGATG AGTCCTTTGA TCCTCTTTAA3644AGGACATAAA AGCTTTATGC AAGAACCAGT GCTGTAAAGT TATAGAATTT CTTTTGCTAT3704ATATGACATT CGACAGAACC TGTTCCGGTT CATCGA3740SPS 2 sequence (Seq. ID No. 2)ATTTTTTTCT CTAAGTTCTC TCTCGCTGTC CTTATCATTT CACCACCTCC ATAAATCTAG60AAACATCTTT TCTACTCCGT TAATCTCTCT AGCACACGGC GGAGGAGTGC GGCGGAGGAG120ATG GCG GGA AAC GAT TGG ATT AAC AGT TAC TTA GAG GCG ATA CTG165Met Ala Gly Asn Asp Trp Ile Asn Ser Tyr Leu Glu Ala Ile Leu1 5 10 15GAT GTT GGA CCA GGG CTA GAT GAT AAG AAG TCA TCG TTG TTG TTG210Asp Val Gly Pro Gly Leu Asp Asp Lys Lys Ser Ser Leu Leu Leu 20 25 30AGA GAA AGA GGG AGG TTT AGT CCG ACG AGG TAC TTT GTT GAG GAA255Arg Glu Arg Gly Arg Phe Ser Pro Thr Arg Tyr Phe Val Glu Glu 35 40 45GTT ATT ACT GGA TTC GAT GAG ACT GAT TTG CAT CGT TCG TGG ATC300Val Ile Thr Gly Phe Asp Glu Thr Asp Leu His Arg Ser Trp Ile 50 55 60CGA GCA CAA GCT ACT CGG AGT CCG CAG AGA AGG AAT ACT AGG CTC345Arg Ala Gln Ala Thr Arg Ser Pro Gln Arg Arg Asn Thr Arg Leu 65 70 75GAG AAT ATG TGC TGG AGG ATT TGG AAT TTG GCT CGC CAG AAA AAG390Glu Asn Met Cys Trp Arg Ile Trp Asn Leu Ala Arg Gln Lys Lys 80 85 90CAG CTT GAG GGA GAG CAA GCT CAG TGG ATG GCA AAA CGC CGT CAA435Gln Leu Glu Gly Glu Gln Ala Gln Trp Met Ala Lys Arg Arg Gln 95 100 105GAA CGT GAA AGA GGT CCC AGA GAA GCA GTT GCT GAT ATG TCA GAG480Glu Arg Glu Arg Gly Arg Arg Glu Ala Val Ala Asp Met Ser Glu 110 115 120GAT CTA TCT GAG GGA GAG AAA GGA GAT ATA GTC GCT GAC ATG TCA525Asp Leu Ser Glu Gly Glu Lys Gly Asp Ile Val Ala Asp Met Ser 125 130 135TCT CAT GGT GAA AGT ACC AGA GGC CGA TTG CCT AGA ATC AGT TCT570Ser His Gly Glu Ser Thr Arg Gly Arg Leu Pro Arg Ile Ser Ser 140 145 150GTT GAG ACA ATG GAA GCA TGG GTC AGT CAG CAG AGA GGA AAG AAG615Val Glu Thr Met Glu Ala Trp Val Ser Gln Gln Arg Gly Lys Lys 155 160 165CTT TAT ATC GTG CTT ATA ACT TTA CAT GGT TTA ATT CGG GGT GAG660Leu Tyr Ile Val Leu Ile Ser Leu His Gly Leu Ile Arg Gly Glu 170 175 180AAT ATG GAG CTT GGA CGG GAT TCT GAT ACT GGT GGT CAG GTG AAG705Asn Met Glu Leu Gly Arg Asp Ser Asp Thr Gly Gly Gln Val Lys 185 190 195TAT GTT GTT GAA CTT GCG AGG GGC TTA GGG TCG ATG CCA GGT GTA750Tyr Val Val Glu Leu Ala Arg Ala Leu Gly Ser Met Pro Gly Val 200 205 210TAT CGG GTT GAC TTG CTT ACT AGA CAA GTA TCT TCA CCA GAA GTA795Tyr Arg Val Asp Leu Leu Thr Arg Gln Val Ser Ser Pro Glu Val 215 220 225GAT TGG AGC TAT GGT GAG CCG ACA GAG ATG CTG ACG CCA ATA AGT840Asp Trp Ser Tyr Gly Glu Pro Thr Glu Met Leu Thr Pro Ile Ser 220 235 240ACA GAC GGC TTG ATG ACT GAG ATG GGG GAG AGT AGT GGT GCT TAT885Thr Asp Gly Leu Met Thr Glu Met Gly Glu Ser Ser Gly Ala Tyr 245 250 255ATT ATT CGC ATT CCT TTT GGA CCA AGA GAG AAA TAT ATT CCA AAA930Ile Ile Arg Ile Pro Phe Gly Pro Arg Glu Lys Tyr Ile Pro Lys 260 265 270GAA CAG CTA TGG CCC TAT ATT CCC GAA TTT GTT GAT GGT GCA CTT975Glu Gln Leu Trp Pro Tyr Ile Pro Glu Phe Val Asp Gly Ala Leu 275 280 285AAC CAT ATT ATT CAA ATG TCC AAA GTT CTT GGG GAG CAA ATT GGT1020Asn His Ile Ile Gln Met Ser Lys Val Leu Gly Glu Gln Ile Gly 290 295 300AGT GGC TAT CCT GTG TGG CCT GTT GCC ATA CAC GGA CAT TAT GCT1065Ser Gly Tyr Pro Val Trp Pro Val Ala Ile His Gly His Tyr Ala 305 310 315GAT GCT GGC GAC TCA GCT GCT CTC CTG TCA GGT GCT TTA AAT GTA1110Asp Ala Gly Asp Ser Ala Ala Leu Leu Ser Gly Ala Leu Asn Val 320 330 335CCA ATG CTT TTC ACT GGT CAC TCA CTT GGT AGA GAT AAG TTG GAG1155Pro Met Leu Phe Thr Gly His Ser Leu Gly Arg Asp Lys Leu Glu 340 345 350CAA CTG TTG GCA CAA GGT CGA AAG TCA AAG GAT GAA ATA AAC TCA1200Gln Leu Leu Ala Gln Gly Arg Lys Ser Lys Asp Glu Ile Asn Ser 355 360 365ACC TAC AAG ATA ATG CGG AGA ATA GAG GCT GAA GAA TTA ACT CTT1245Thr Tyr Lys Ile Met Arg Arg Ile Glu Ala Glu Glu Leu Thr Leu 370 375 380GAT GCT TCC GAA ATT GTC ATC ACT AGT ACA AGA CAG GAG ATT GAC1290Asp Ala Ser Glu Ile Val Ile Thr Ser Thr Arg Gln Glu Ile Asp 385 390 395GAG CAA TGG CGT TTG TAT GAT GGG TTT GAT CCA ATA TTA GAG CGT1335Glu Gln Trp Arg Leu Tyr Asp Gly Phe Asp Pro Ile Leu Glu Arg 400 405 410AAG TTA CGT GCA AGG ATC AAG CGC AAT GTC AGC TGT TAT GGC AGG1380Lys Leu Arg Ala Arg Ile Lys Arg Asn Val Ser Cys Tyr Gly Arg 415 420 425TTT ATG CCT CGT ATG GCT GTA ATT CCT CCT GGG ATG GAG TTC CAC1425Phe Met Pro Arg Met Ala Val Ile Pro Pro Gly Met Glu Phe His 430 435 440CAT ATT GTG CCA CAT GAA GGT GAC ATG GAT GGT GAA ACA GAA GGA1470His Ile Val Pro His Glu Gly Asp Met Asp Gly Glu Thr Glu Gly 445 450 455AGT GAA GAT GGG AAG ACC CCC GAT CCA CCT ATT TGG GCA GAG ATT1515Ser Glu Asp Gly Lys Thr Pro Asp Pro Pro Ile Trp Ala Glu Ile 460 465 470ATG CGC TTC TTT TCT AAT CCA AGG AAG CCT ATG ATA CTC GCA CTT1560Met Arg Phe Phe Ser Asn Pro Arg Lys Pro Met Ile Leu Ala Leu 475 480 485GCT AGG CCT GAT CCC AAG AAG AAC CTC ACT ACT TTA GTG AAA GCA1605Ala Arg Pro Asp Pro Lys Lys Asn Leu Thr Thr Leu Val Lys Ala 490 495 500TTT GGT GAA TGT CGT CCA TTG AGA GAG CTT GCT AAT CTT ACT TTG1650Phe Gly Glu Cys Arg Pro Leu Arg Glu Leu Ala Asn Leu Thr Leu 505 510 515ATA ATG GGT AAT CGA GAT AAT ATC GAC GAA ATG TCT AGC ACC AAT1695Ile Met Gly Asn Arg Asp Asn Ile Asp Glu Met Ser Ser Thr Asn 520 525 520TCT GCA CTT CTT CTT TCA ATC TTG AAA ATG ATA GAT AAG TAT GAT1740Ser Ala Leu Leu Leu Ser Ile Leu Lys Met Ile Asp Lys Tyr Asp 525 540 540CTT TAT GGT CAA GTA GCT TAT CCT AAA CAC CAC AAG CAG TCA GAT1785Leu Tyr Gly Gln Val Ala Tyr Pro Lys His His Lys Gln Ser Asp 545 550 555GTT CCT GAT ATC TAC CGT CTT GCT GCA AAG ACT AAG GGT GTT TTT1830Val Pro Asp Ile Tyr Arg Leu Ala Ala Lys Thr Lys Gly Val Phe 560 565 570ATT AAT CCA GCT TTT ATT GAG CCT TTT GGA CTG ACT TTG ATT GAG1875Ile Asn Pro Ala Phe Ile Glu Pro Phe Gly Leu Thr Leu Ile Glu 575 580 585GCA GCA GCT TAT GGT CTC CCA ATG GTA GCC ACA AAA AAT GGA GGA1920Ala Ala Ala Tyr Gly Leu Pro Met Val Ala Thr Lys Asn Gly Gly 590 595 600CCT GTT GAT ATA CAT AGG GTT CTT GAC AAT GGT CTC TTA GTG GAT1965Pro Val Asp Ile His Arg Val Leu Asp Asn Gly Leu Leu Val Asp 605 610 615CCC CAT GAT CAG CAG GCA ATT GCT GAT GCT CTT TTG AAG TTG GTT2010Pro His Asp Gln Gln Ala Ile Ala Asp Ala Leu Leu Lys Leu Val 620 625 630GCT GAT AAG CAA CTG TGG GCT AAA TGC AGG GCA AAT GGA TTA AAA2055Ala Asp Lys Gln Leu Trp Ala Lys Cys Arg Ala Asn Gly Leu Lys 635 640 645AAT ATC CAC CTT TTC TCA TGG CCC GAG CAC TGT AAA ACT TAT CTA2100Asn Ile His Leu Phe Ser Trp Pro Glu His Cys Lys Thr Tyr Leu 650 655 660TCC CGG ATA GCT AGC TGC AAA CCA AGG CAA CCA CGC TGG CTG AGA2145Ser Arg Ile Ala Ser Cys Lys Pro Arg Gln Pro Arg Trp Leu Arg 665 670 675TCC ATT GAT GAT GAT GAT GAA AAT TCA GAA ACA GAT TCA CGT AGT2190Ser Ile Asp Asp Asp Asp Glu Asn Ser Glu Thr Asp Ser Pro Ser 680 685 690GAT TCC TTG AGA GAT ATT CAT GAT ATA TCT CTG AAT TTG AGA TTT2235Asp Ser Leu Arg Asp Ile His Asp Ile Ser Leu Asn Leu Arg Phe 695 700 705TCA TTA GAT GGG GAA AAG AAT GAC AAT AAA GAA AAT GCT GAT AAT2280Ser Leu Asp Gly Glu Lys Asn Asp Asn Lys Glu Asn Ala Asp Asn 710 715 720ACA TTA GAC CCC GAA GTT CGA AGG AGG AAG TTA GAG AAT GCT GTT2325Thr Leu Asp Pro Glu Val Arg Arg Ser Lys Leu Glu Asn Ala Val 725 730 735TTG TCC TTA TCT AAG GGT GCA CTG AAG AGC ACA TCA AAA TCT TGG2370Leu Ser Leu Ser Lys Gly Ala Leu Lys Ser Thr Ser Lys Ser Trp 740 745 750TCG TCA GAC AAG GCA GAC CAA AAC CCT GGT GCT GGT AAA TCC CCA2415Ser Ser Asp Lys Ala Asp Gln Asn Pro Gly Ala Gly Lys Phe Pro 755 760 765GCG ATT AGG AGG AGG CGA CAT ATT TTT GTT ATT GCA GTG GAT TGT2460Ala Ile Arg Arg Arg Arg His Ile Phe Val Ile Ala Val Asp Cys 770 775 780GAT GCT AGC TCA GGA CTC TCT GGA AGT GTG AAA AAG ATA TTT GAG2505Asp Ala Ser Ser Gly Leu Ser Gly Ser Val Lys Lys Ile Phe Glu 785 790 795GCT GTA GAG AAG GAA AGG GCA GAG GGT TCC ATT GGA TTT ATC CTG2550Ala Val Glu Lys Gla Arg Ala Glu Gly Ser Ile Gly Phe Ile Leu 800 805 810GCT ACA TCT TTC AAT ATA TCA GAA GTA CAG TCT TTC CTG CTT TCA2595Ala Thr Ser Phe Asn Ile Ser Glu Val Gln Ser Phe Leu Leu Ser 815 820 825GAG GGC ATG AAT CCT ACT GAT TTT GAT GCT TAG ATA TGC AAT AGT2640Glu Gly Met Asn Pro Thr Asp Phe Asp Ala Tyr Ile Cys Asn Ser 830 835 840GGT GGT GAT CTT TAT TAT TCG TCC TTC CAT TCT GAG CAA AAT CCT2685Gly Gly Asp Leu Tyr Tyr Ser Ser Phe His Ser Glu Gln Asn Pro 845 850 855TTT GTA GTT GAC TTG TAC TAT CAC TCA CAT ATT GAG TAT CGT TGG2730Phe Val Val Asp Leu Tyr Tyr His Ser His Ile Glu Tyr Arg Trp 860 865 870CGG GGC GAA GGA TTG AGA AAG ACT TTG GTG CGT TGG GGC GCC TCT2775Gly Gly Glu Gly Leu Arg Lys Thr Leu Val Arg Trp Ala Ala Ser 875 880 885ATC ATT GAT AAG AAT GGT GAA AAT GGA GAT CAC ATT GTT GTT GAG2820Ile Ile Asp Lys Asn Gly Glu Asn Gly Asp His Ile Val Val Glu 890 895 900GAT GAA GAC AAT TCA GCT GAC TAC TGC TAT ACT TTC AAA GTG TGC2865Asp Glu Asp Asn Ser Ala Asp Tyr Cys Tyr Thr Phe Lys Val Cys 905 910 915AAG CCT GGG AGG GTT GCT CCA TGT AAA GAG GTT AGA AAA GTA ATG2910Lys Pro Gly Thr Val Pro Pro Ser Lys Glu Leu Arg Lys Val Met 920 925 930CGA ATT CAG GCA GTT CGT TGT CAC GCT GTT TAT TGT GAA AAT GGG2955Arg Ile Gln Ala Leu Arg Cys His Ala Val Tyr Cys Gln Asn Gly 935 940 945ACT AGG ATT AAT GTG ATG CGT GTA GTG GCA TGT GGG TGG GAA GCA3000Ser Arg Ile Asn Val Ile Pro Val Leu Ala Ser Arg Ser Gln Ala 950 955 960GTG AGG TAG TTA TAT GTG GGA TGG GGA ATG GAG TTG TGG AAG TTG3045Leu Arg Tyr Leu Tyr Leu Arg Trp Gly Met Asp Leu Ser Lys Leu 965 970 975GTG GTT TTC GTG GGA GAA ACT GGT GAT ACG GAT TAT GAA GGA TTA3090Val Val Phe Val Gly Glu Ser Gly Asp Thr Asp Tyr Glu Gly Leu 980 985 990ATG GGT GGT GTA GGG AAG GGT GTG ATA ATG AAA GGG GTG TGG ACT3135Ile Gly Gly Leu Arg Lys Ala Val Ile Met Lys Gly Leu Cys Thr 995 1000 1005AAT GCA AGC AGC TTA ATT GAG GGT AAT AGG AAT TAG CGG GTA TGT2180Asn Ala Ser Ser Leu Ile His Gly Asn Arg Asn Tyr Pro Leu Ser 1010 1015 1020GAT GTT TTA CGA TTC GAG AGG GCT AAT GTG ATG CAA GGC GAG GAG3225Asp Val Leu Pro Phe Asp Ser Pro Asn Val Ile Gln Ala Asp Glu 1025 1030 1035GAA TGT AGC AGG ACC GAA ATG CGT TGC TTA CTG GAG AAA GTA GGG2270Glu Cys Ser Ser Thr Glu Ile Arg Cys Leu Leu Glu Lys Leu Ala 1040 1045 1050GTA CTC AAA GGA TAA TACCGTTGGC CGTTTGATTG TCAAAAACCT3315Val Leu Lys Gly End 1054ATATGAGCTA TAAGACTATG GCATGAAAAG AATGGCCATC CATTTGGCTT GTCTTTTGAA2375GCTGTTAATA GTTTTGAAGA GAGTACAAAA TGAGATGAGT CCTTTGATCG TCTTTAAAGG3435ACATAAAAGC TTTATGCAAG AAGCAGTGCT GTAAAGTTAT AGAATTTCTT TTGCTATATA3495TGACATTCGA CAGAACCAGT TCGGGTTCAT GGAGAAAAAG AAATAAATTT GAAGTTATAA3555ACATGCCTGA TCATGTAAAT TATGATATAC ATCCATGGGA AGGGATTATG GATGGGTTAT3615CAGATTTTTT3625SPS 3 sequence (Seq. ID No.3) ATTTTTT TCTCTAAATT GTGTGTGACT GTGCTTATGA TTTCACGACC TCGATAAATC57TAGAAACATC TTTTCTATTC CGTTAATCTC TGTAGCACAC GGCGGAGTGC GGCGGAGGAG117ATG GCG GGA AAG GAC TGG ATT AAC AGT TAG TTA GAG GCG ATA CTG162Met Ala Gly Asn Asp Trp Ile Asn Ser Tyr Leu Glu Ala Ile Leu1 5 10 15GAT GTA GGA CCA GGG CTA CAT GAT AAG AAA TCA TCG TTG TTG TTG207Asp Val Gly Pro Gly Leu Asp Asp Lys Lys Ser Ser Leu Leu Leu 20 25 30AGA GAA AGA GGG AGG TTT AGT CCG ACG AGG TAG TTT GTT GAG GAA252Arg Glu Arg Gly Arg The Ser Pro Thr Arg Tyr The Val GTh Glu 25 40 45GTT ATT ACT GGA TTC CAT GAG ACT GAT TTG CAT CCC TCG TGG ATG297Val Ile Thr Gly phe Asp Glu Thr Asp Leu His Arg Ser Trp Ile 50 55 60GGA GCA CAA GCT ACT CGG ACT CCG GAG GAG AGG AAT ACT AGG CTC342Arg Ala Gln Ala Thr Arg Ser Pro Gln Glu Arg Asn Thr Arg Leu 65 70 75GAG AAT ATG TGC TGG AGC ATT TGG AAT TTG GCT CGC GAG AAA AAG287Glu Asn Met Cys Trp Arg Ile Trp Asn Leu Ala Arg Gln Lys Lys 80 85 90GAG CTT GAG GGA GAG CAA CCT GAG TGG ATG CCA AAA CGG CGT GAA422Gln Leu Glu Gly Glu Gln Ala Gln Trp Met Ala Lys Arg Arg Gln 95 100 105GAA CGT GAG AGA GGT CCC AGA GAA GGA CTT GCT CAT ATG TCA GAG477Glu Arg Glu Arg Gly Arg Arg Glu Ala Val Ala Asp Met Ser Glu 110 115 120CAT CTA TCT GAG GGA GAG AAA GGA CAT ATA GTC GCT GAG ATG TCA522Asp Leu Ser Glu Gly Glu Lys Gly Asp Ile Val Ala Asp Met Ser 125 130 135TCT CAT GCT GAA AGT ACC AGA GGC CGA TTG GCT AGA ATG AGT TGT567Ser His Gly Glu Ser Thr Arg Gly Arg Leu Pro Arg Ile Ser Ser 140 145 150GTT GAG ACA ATG GAA GCA TGG GTC AGT CAG CAG AGA GCA AAG AAG612Val Glu Thr Met Glu Ala Trp Val Ser Gln Gln Arg Gly Lys Lys 155 160 165GTT TAT ATG GTG GTT ATA AGT TTA CAT GGT TTA ATT GGG GCT GAG657Leu Tyr Ile Val Leu Ile Ser Leu His Gly Leu Ile Arg Gly Glu 170 175 180AAT ATG GAG CTT GGA CGG GAT TGT GAT ACT GCT GGT CAG GTG AAG702Asn Met Glu Leu Gly Arg Asp Ser Asp Thr Gly Gly Gln Val Lys 185 190 195TAT GTA GTT GGA GCA ACT GTT GCA CAA GGT CGT TTG TCA AAG GAT747Tyr Val Val Gly Ala Thr Val Ala Gln Gly Arg Leu Ser Lys Asp 200 205 210GAA ATA AAC TCA ACG TAG AAG ATA ATG CGG AGA ATA GAG GGT GAA792Glu Ile Asn Ser Thr Tyr Lys Ile Met Arg Arg Ile Glu Ala Glu 215 220 225GAA TTA ACT CTT GAT GCT TGG GAA ATT GTG ATC ACT AGT ACA AGA837Glu Leu Thr Leu Asp Ala Ser Glu Ile Val Ile Thr Ser Thr Arg 230 235 240CAG GAG ATT GAC GAG CAA TGG CGT TTG TAT GAT GGG TTT GAT GCA882Gln Glu Ile Asp Glu Gln Trp Arg Leu Tyr Asp Gly Phe Asp Pro 245 250 255ATA TTA GAG CGT AAG TTA GCT GCA AGG ATG AAG CGC AAT GTG AGG927Ile Leu Glu Arg Lys Leu Arg Ala Arg Ile Lys Arg Asn Val Ser 260 265 270TGT TAT GGC AGG TTT ATG CCT CGT ATG GGT GTA ATT CGT CCT GGG972Cys Tyr Gly Arg Phe Met Pro Arg Met Ala Val Ile Pro Pro Gly 275 280 285ATG GAG TTC CAC CAT ATT GTG CCA CAT GAA GGT GAC ATG GAT GGT1017Met Glu Phe His His Ile Val Pro His Glu Gly Asp Met Asp Gly 290 295 300GAA ACA GAA GGA AGT GAA GAT GGA AAG ACC CGG GAT CGA GCT ATT1062Glu Thr Glu Gly Ser Glu Asp Gly Lys Thr Pro Asp Pro Pro Ile 305 310 315TGG GCA GAG ATT ATG CGC TTG TTT TGT AAT GCA AGG AAG GCT ATG1107Trp Ala Glu Ile Met Arg Phe Phe Ser Asn Pro Arg Lys Pro Met 320 330 335ATA GTG GCA GTT GCT AGG CGT GAT CGG AAG AAG AAG GTG ACT ACT1152Ile Leu Ala Leu Ala Arg Pro Asp Pro Lys Lys Asn Leu Thr Thr 340 345 350TTA GTG AAA GCA TTT GGT GAA TGT CGT GCA TTG AGA GAG CTT GCT1197Leu Val Lys Ala Phe Gly Glu Cys Arg Pro Leu Arg Asp Leu Ala 355 360 365AAT GTT ACT TTG ATA ATG GGT AAT GGA GAT AAT ATG GAG GAA ATG1242Asn Leu Thr Leu Ile Met Gly Asn Arg Asp Asn Ile Asp Glu Met 370 375 380TGT AGG ACC AAT TGT GCA GTT GTT GTT TGA ATG TTG AAG ATG ATA1287Ser Ser Thr Asn Ser Ala Leu Leu Leu Ser Ile Leu Lys Met Ile 385 390 395GAT AAG TAT GAT GTT TAT GGT GTA GTA GCT TAT GCT AAA CAC CAC1332Asp Lys Tyr Asp Leu Tyr Gly Leu Val Ala Tyr Pro Lys His His 400 405 410AAG CAG TCA GAT GTT CGT GAT ATG TAC CGT GTT GGT CCA AAG ACT1377Lys Gln Ser Asp Val Pro Asp Ile Tyr Arg Leu Ala Ala Lys Thr 415 420 425AAG GGT GTT TTT ATT AAT CGA GCT TTT ATT GAG CGT TTT GGA CTG1422Lys Gly Val Phe Ile Asn Pro Ala Phe Ile Glu Pro Phe Gly Leu 430 435 440ACT TTG ATT GAG GGA GGA GCT TAT GGT GTG GCA ATG GTA GCC ACA1467Thr Leu Ile Glu Ala Ala Ala Tyr Gly Leu Pro Met Val Ala Thr 445 450 455AAA AAT GGA GGA CGT GTT GAT ATA GAT AGG GTT GTT GAG AAT GGT1512Lys Asn Gly Gly Pro Val Asp Ile His Arg Val Leu Asp Asn Gly 460 465 470GTG TTA GTG GAT CGG GAT GAT CAG CAG GCA ATT GGT GAT GCT CTT1557Leu Leu Val Asp Pro His Asp Gln Gln Ala Ile Ala Asp Ala Leu 475 480 485TTG AAG TTG GTT GCT GAT AAG GAA GTG TGG GCT AAA TGG AGG GCA1602Leu Lys Leu Val Ala Asp Lys Gln Leu Trp Ala Lys Cys Arg Ala 490 495 500AAT GGA TTA AAA AAT ATG CAC GTT TTC TCA TGG CCC GAG CAC TGT1647Asn Gly Leu Lys Asn Ile His Leu Phe Ser Trp Pro Glu His Cys 505 510 515AAA ACT TAT GTA TGG CGG ATA GCT AGG TGG AAA CGG AGG CAA CAT1692Lys Thr Tyr Leu Ser Arg Ile Ala Ser Cys Lys Pro Arg Gln His 520 525 530TGG TTG AGA GAT ATT GAT GAT ATA TGT GTG AAT TTG AGA TTT TGA1727Ser Leu Arg Asp Ile His Asp Ile Ser Leu Asn Leu Arg Phe Ser 535 540 540TTA GAT GGG GAA AAG AAT GAC AAT AAA GAA AAT GCT GAT AAT ACA1782Leu Asp Gly Glu Lys Asn Asp Asn Lys Glu Asn Ala Asp Asn Thr 545 550 555TTA GAC CCC GAA GTT CGA AGG AGC AAG TTA GAG AAT GCT GTT TTG1827Leu Asp Pro Glu Val Arg Arg Ser Lys Leu Glu Asn Ala Val Leu 560 565 570TCC TTA TCT AAG GGT GCA CTG AAG AGC ACA TCA AAA TCT TGG TCG1372Ser Leu Ser Lys Gly Ala Leu Lys Ser Thr Ser Lys Ser Trp Ser 575 530 585TCA GAC AAG GCA GAC CAA AAT CCT GGT GCT GGT AAA TTC CCA GCG1917Ser Asp Lys Ala Asp Gln Asn Pro Gly Ala Gly Lys Phe Pro Ala 590 595 600ATT AGG AGG AGG CGA CAT ATT TTT GTT ATT GCA GTG GAT TGT GAT1962Ile Arg Arg Arg Arg His Ile Phe Val Ile Ala Val Asp Cys Asp 605 610 615GCT AGC TCA GGA CTC TCT GGA AGT ATG AAA AAG ATA TTT GAG GCT2007Ala Ser Ser Gly Leu Ser Gly Ser Met Lys Lys Ile Phe Glu Ala 620 625 630GTA GAG AAG GAA AGG GCA GAG GGT TCC ATT CGA TTT ATC CTT GCT2052Val Glu Lys Glu Arg Ala Glu Gly Ser Ile Gly Phe Ile Leu Ala 635 640 645ACA TCT TTC AAT ATA TCA GAA GTA CAG TCT TTC CTG CTT TCA GAG2097Thr Ser Phe Asn Ile Ser Glu Val Glu Ser Phe Leu Leu Ser Glu 650 655 660GGC ATG AAT CGT ACT GAG CAA AAT CCT TTT GTA GTT GAC TTG TAC2142Gly Met Asn Pro Thr Glu Gln Asn Pro Phe Val Val Asp Leu Tyr 665 670 675TAT CAC TCA CAT ATT GAG TAT CGT TGG GGG GGC GAA GGG TTG AGA2187Tyr His Ser His Ile Glu Tyr Arg Trp Gly Gly Glu Gly Leu Arg 680 685 690AAG ACT TTG GTG CGT TGG 0CC 0CC TCT ATC ATT GAT AAG AAT GGT2222Lys Thr Leu Val Arg Trp Ala Ala Ser Ile Ile Asp Lys Asn Gly 695 700 705GAA AAT GGA GAT CAC ATT GTT GTT GAG GAT GAA GAC AAT TCA GCT2277Glu Asn Gly Asp His Ile Val Val Glu Asp Glu Asp Asn Ser Ala 710 715 720GAC TAC TGC TAT ACA TTC AAA GTT TGC AAG CCT GGG ACG GTT CCT2222Asp Tyr Cys Tyr Thr Phe Lys Val Cys Lys Pro Gly Thr Val Pro 725 730 725CCA TCT AAA GAA CTT AGA AAA GTA ATG CGA ATT CAG GCA CTT CGT 2267Pro Ser Lys Glu Leu Arg Lys Val Met Arg Ile Gln Ala Leu Arg 740 745 750TGT CAC GCT GTT TAT TGT CAA AAT GGG AGT AGG ATT AAT GTG ATC2412Cys His Ala Val Tyr Cys Gln Asn Gly Ser Arg Ile Asn Val Ile 755 760 765CCT GTA CTG GCA TCT CGG TCC CAA GCA CTC AGG TAC TTA TAT CTG2457Pro Val Leu Ala Ser Arg Ser Gln Ala Leu Arg Tyr Leu Tyr Leu 770 775 780CGA TGG GGA ATG GTC CCT GTA CTG GCA TCT CGG TCC CAA GCA CTC2502Arg Trp Gly Met Val Pro Val Leu Ala Ser Arg Ser Gln Ala Leu 785 790 795AGG TAC TTA TAT CTG CGA TGG GGA ATG GTC CCT GTA CTG GCA TCT2547Arg Tyr Leu Tyr Leu Arg Trp Gly Met Val Pro Val Leu Ala Ser 800 805 810CGG TCC CAA GCA CTC AGG TAC TTA TAT GTG CGA TGG CCA ATG GAC2592Arg Ser Gln Ala Leu Arg Tyr Leu Tyr Leu Arg Trp Gly Met Asp 815 820 825TTG TCG AAG TTG GTG GTT TTC GTC CGA GAA ACT CGT GAT ACC CAT2627Leu Ser Lys Leu Val Val Phe Val Gly Glu Ser Gly Asp Thr Asp 830 835 840TAT GAA CCA TTG ATC GGT GCT CTA CGC AAG GCT GTC ATA ATG AAA2682Tyr Glu Gly Leu Ile Gly Cly Leu Arg Lys Ala Val Ile Met Lys 845 850 855CGA CTC TGC ACT AAT GCA AGC AGC TTA ATT CAC GGT AAT AGG AAT2727Gly Leu Cys Thr Asn Ala Ser Ser Leu Ile His Gly Asn Arg Asn 860 865 870TAC CCC CTA TCT CAT GTT TTA CCA TTC GAG ACC CCT AAT GTC ATC2772Tyr Pro Leu Ser Asp Val Leu Pro Phe Glu Ser Pro Asn Val Ile 875 880 885CAA CCG CAT GAG GAA TGT AGC ACC ACC CGA ATC CGT TCC TTA CTG2817Gln Ala Asp Glu Glu Cys Ser Ser Thr Gly Ile Arg Ser Leu Leu 905 910 915GAG AAA CTA CCC GTA CTC AAA CCA TAA TACCCTTCCC CCTTTCATTG2864Glu Lys Leu Ala Val Leu Lys Gly End 920TCAAAAACCT ATATGAGCTA AGATTATCCC ATGAAAAGAA TGCCCATCCA TTTCCCTTCT2924CTTTTG2930

[0030] All sequences are cDNA sequences and stem from a cDNA libaray of leaf tissue. The expression gene is the same in various plant tissues. As promoter, there can generally be used any promoter which is active in plants. The promoter should ensure that the foreign gene is expressed in the plant. The promoter can be so chosen that the expression occurs only in specified tissues, at a determined time point in the plant's development or at a time point determined by outside influences. The promoter can be homologous or heterologous to the plant. Suitable promoters are e.g. the promoter of the 355 RNA of the cauliflower mosaic virus, the patatin promoter B33 (Rocha-Sosa et al. (1989) EMBO J 8: 23-29) or a promoter that ensures an expression only in photosynthetically active tissues. Other promoters can be used which ensure an expression only in specified organs, such as the root, tuber, seed, stem or specified cell types such as mesophyllic, epidermal or transport cells. For hindering cold sweetening, suitable promoters are those which ensure an activation of the transcription is stored in harvested parts of the plants. For this, there can be considered cold induced promoters or such promoters, that become active during the transition of the tuber from the phase where it stores material to the phase where it gives up material.

[0031] The coding sequence contains the information for the formation of an mRNA for the sucrose-phosphate-synthase or for the formation of an anti-sense RNA for the SPS. whether the translatable MRNA or an anti-sense RNA is formed, depends on the orientation of the coding sequence in relation to the promoter. If the 3′ end of the coding sequence is fused to the 3′ end of the promoter, an anti-sense RNA results, and by fusion of the 5′ end of the coding to the 3′ end of the promoter a translatable RNA results. This latter leads to an increase of the SPS activity in the cell, whilst the first leads to a reduction of the SPS activity in the cell. Such a reduction of SPS activity is of especial significance in view of the undesirable formation of sucrose and/or reducing sugars as a result of cold storage of harvested organs.

[0032] The coding sequence for SPS can be one of the three described above or one that is derived by modification the sequences described above. A derivation can be out, e.g. by current methods of mutagenesis and/or recombination. For this especially, changes of SPS sequences are envisaged, that lead to a neutralization of the plant's own regulation mechanism.

[0033] The DNA sequences of the invention can be used for the preparation of derivatives whose gene products are not subjected to the plant's own activity regulation during a phosphorylation reaction.

[0034] Further, the sequences can also be used for the preparation of derivatives by targeted and non-targeted mutagenesis.

[0035] The invention relates further to derivatives of the DNA sequences of the invention that are-obtained by exchange of single bases or by deletion or insertion of base sequences and which code for proteins with a comparable activity to sucrose-phosphate-synthase.

[0036] The 5′ untranslated area of the sequence Seq. ID No 1 definitely does not belong to SPS, but is added as a cloning artefact. The methionine start codon of the coding region lies in a region in which no homology of the amino acid sequence to the other SPS sequences is involved. Since this sequence does not also fully coincide in the homologous region with one of the other sequences, it is recognisable that the sequence Seq. ID No 1 is not a derivative of the sequences Seq. ID No 2 and Seq. ID No 3.

[0037] The termination sequence provides the correct finishing of the transcription and the attachment of a polyadenyl group to the RNA. This polyadenyl group has an important function in the stabilisation of RNA molecules in the cells. With suitable plasmids, which contain the DNA sequences of the invention, plants can be transformed with the object of raising and/or reducing the SPS activity and/or the modification of the sucrose concentration.

[0038] Plasmids, that can be used are e.g. p35S-anti-pot-SPS (DSM 7125) and pB33-anti-pot-SPS (DSM 7124). With the gene 35S-anti-pot-SPS, located on the plasmid p355-anti-pot-SPS, the concentration of the mRNA for the SPS protein and the enzymatic activity, for example, can be reduced. With the gene B335-anti-pot-SPS, located on the plasmid pB33-anti-pot-SPS, the concentration of the mRNA for the SPS protein and the enzymatic activity, specifically for potato tubers for example, can be reduced. In a similar way to the SPS sequence (Seq. ID No. 1) located on this plasmid, other SPS sequences, e.g. the sequences Seq. ID No. 2 and Seq. ID No. 3 also be cloned in suitable vectors and for the same purpose.

[0039] In the plant, the SPS is subjected to an activity control by phosphorylation. This allows the plant to regulate the activity of the enzyme within a fixed frame independent of the amount of the SPS protein. If one of the changes occurring outside the activity of the SPS is to achieved, it is necessary to evade the plant's own regulation mechanism. Therefore changing the phosphorylation possibilities is an important target for influencing the SPS activity and thus the sucrose content of the plant.

[0040] It is not known, in which position in the SPS protein, target directed changes of the coding regions can be achieved, which serve the purpose of introducing in the plant, SPS activity, which is not subject to any of the plant's own controls.

[0041] The DNA sequence described here, which contains the coding region for SPS from Solanum tuberosum, allows the identification of the sites of protein phosphorylation of the SPS. By using standard methods (Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A laboratory Manual, 2nd. Edn., Cold Spring Harbor Laboratory Press, NY, USA), a localisation of the phosphorylation positions of SPS is possible using the DNA sequences of the invention. These being known, by use of the plasmids with the SPS sequence, a target directed mutagenesis (Sambrook et al, 1989) of the coding region of SPS and/or a non-target directed mutagenesis (Sambrook et al, 1989) and subsequent probing of the desired mutations of the coding region of the SPS can be undertaken. Derivatives of the coding region can be prepared with the help of this plasmid, whose derived proteins are not subjected to the plants own regulation mechanisms.

[0042] Since the SPS enzyme is regulated by phosphorylation in all tested species, except maize, one can refer to sequence comparisons, to identify possible phosphorylation sites. The criterium for this is that a serine residue appears in an acidic medium in the regulated SPS protein, but not however with maize. There are 12 such serine residues in the sequences Sea. ID No. 2 and Seq ID No. 3. In the sequence Seq ID No. 1, the first of the 12 serine residues is missing, since the coding region begins just later. The sequence Seq. ID No. 1 is thus especially suitable for the production of an SPS activity in plants, that is not liable to endogenous activity regulation.

[0043] For the introduction of the SPS sequence in higher plants, a large number of cloning vectors are available, which contain a replication signal for E. coli and a marker, which allows a selection of the transformed cells. Examples of vectors are pBR 322, pUC-series, M13 mp-series, PACYC 184; EMBL 3 etc. According to the introduction method of the desired gene in the plant, other DNA sequences may be suitable. Should the Ti- or Ri-plasmid be used, e.g. for the transformation of the plant cell, then at least the right boundary, often however both the right and left boundary of the Ti- and Ri-Plasmid T-DNA, is attached, as a flanking region, to the gene being introduced. The use of T-DNA for the transformation of plants cells has been intensively researched and is well described in EP 120 516; Hoekama, In: The Binary Plant Vector System, Offset-drukkerij Kanters B. V. Alblasserdam, (1985), Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46 and An et al. (1985) EMBO J. 4: 277-287. Once the introduced DNA is integrated in the genome, it is as a rule stable there and remains also in the offspring of the original transformed cells. It normally contains a selection marker, which induces resistance in the transformed plant cells against a biocide or antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc. The individual marker employed should therefore allow the selection of transformed cells from cells, which lack the introduced DNA.

[0044] For the introduction of DNA into a plant, besides transformation using Agrobacteria, there are many other techniques available. These techniques include the fusion of protoplasts, microinjection of DNA and electroporation, as well as ballistic methods and virus infection. From the transformed plant material, whole plants can be regenerated in a suitable medium, which contains antibiotics or biocides for the selection. The resulting plants can then betested for the presence of introduced DNA. No special demands are placed on the plasmids in injection and electroporation. Simple plasmids, such as e.g. pUC-derivatives can be used. Should however whole plants be regenerated from such transformed cells the presence of a selectable marker gene is necessary. The transformed cells grow within the plants in the usual manner (see also McCormick et al.(1986) Plant Cell Reports 5: 81-84). These plants can be grown normally and crossed with plants, that possess the same transformed genes or different. The resulting hybrid individuals have the corresponding phenotypical properties.

Deposits

[0045] The following plasmids were deposited at the Deutschen Sammlung von Mikroorganismen (DSM) in Braunschweig, Germany on the 12.06.1992 (deposit number):

[0046] Plasmid p35S-anti-pot-SPS (DSM 7125) Plasmid pB33-anti-pot-SPS (DSM 7124)

DESCRIPTION OF THE FIGURES

[0047]
FIG. 1: Structure of the 35S-anti-pot-SPS gene

[0048] A=Fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al.,1980, Cell 21: 285-294)

[0049] B=Fragment B: sucrose phosphate synthase, EcORV Fragment (nt 1 bis 2011), ca. 2000 bp, orientation: anti-sense

[0050] C=Fragment C: nt 11748-11939 of the T-DNA of the Ti-plasmid pTiACH5; Gielen et al., 1984, EMBO J 3: 835-846)

[0051]
FIG. 2: Structure of the B33-anti-pot-SPS gene

[0052] A=Fragment A: B33 promoter of the patatin gene from S. tuberosum, (Rocha-Sosa et al., 1989, EMBO J 8: 23-29), ca 530 bp

[0053] B=Fragment B: sucrose phosphate synthase (s. FIG. 2), EcoRV fragment (nt 2011 bis 1), ca. 2000 bp, orientation: anti-sense

[0054] C=Fragment C: nt 11748-11939 of T-DNA of the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J 3: 835-846)

[0055]
FIG. 3: shows the results of the transformation of transgenic potato plants.

[0056] Control=wild type plants

[0057] 1-75=individual transgenic plants

[0058]
FIG. 4: shows the results of the transformation of potato plants

[0059] Control=wild type plants

[0060] 3-20=individual transgenic plants

[0061] In order to understand the examples forming the basis of this invention all the processes necessary for these tests and which are known per se will first of all be listed:

1. Cloning Process

[0062] The vectors pUC 18/19 and M13mp10 series (Yanisch-Perron et al. (1985) Gene 33: 103-119), as well as the vector EMBL 3 (Frischauf et al. (1983) J Mol Biol 170: 827- 842) were used for cloning.

[0063] For the plant transformations, the gene constructs were cloned in the binary vector BIN 19 (Bevan (1984) Nucl. Acids Res 12: 8711-8720)

2. Bacterial Strains

[0064] The E. coli strain BMH71-18 (Messing et al., Proc. Natl. Acad. Sci. USA (1977), 24, 6342-6346) or TB1 was used for the pUC and M13 mP vectors.

[0065] For the vector BIN19, the E. coli strain TB1 exclusively, was used. TB1 is a recombinant-negative, tetracycline-resistant derivative of strain JM101 (Yanisch-Perron et al., Gene (1985), 33, 103-119). The genotype of the TB1 strain is (Bart Barrel, personal communication): F′ (traD36, proAB, lacI, lacZΔM15), Δ (lac, pro), SupE, this, recA, Sr1::Tn10(TcR).

[0066] The transformation of the plasmids into the potato plants was carried out using Agrobacterium tumefaciens strain LBA4404 (Bevan, (1984), Nucl. Acids Res. 12, 8711-8720).

3. Transformation of Agrobacterium tumefaciens

[0067] In the case of BIN19 derivatives, the insertion of the DNA into the Agrobacterium was effected by direct transformation in accordance with the method of Holsters et al., (1978) (Mol Gene Genet 163: 181-187). The plasmid DNA of the transformed Agrobacterium was isolated in accordance with the method of Birnboim and Doly (1979) (Nucl Acids Res 7: 1513-1523) and was analysed by gel electrophoresis after suitable restriction cleavage.

4. Plant Transformation

[0068] Ten small leaves, wounded with a scalpel, of a sterile potato culture were placed in 10 ml of MS medium with 2% sucrose containing 30-50 μl of an Agrobacterium tumefaciens overnight culture grown under selection. After 3-5 minutes gentle shaking, the leaves were laid out on MS medium of 1.6% glucose, 2 mg/l of zeatin ribose, 0.02 mg/l of naphthylacetic acid, 0.02 mg/l of gibberellic acid, 500 mg/l of claforan, 50 mg/l of kanamycin and 0.8% bacto agar. After incubation for one week at 25° C. and 3000 lux, the claforan concentration in the medium was reduced by half.

5. SPS Activity Test

[0069] The SPS activity was determined according to the method of Siegel and Stitt (1990, Plant Science 66: 205-210) in a two stage analysis. To 180 μl of a solution of 50 mM HEPES/KOH (pH 7.4), 5 mM magnesium chloride, 5 mM fructose-6-phosphate, 25 mM glucose-6-phosphate and 6 mM uridine-5′-diphosphoglucose 20 μl of probe was added and incubated for 10 minutes at 25° C. It was heated for 3 minutes at 95°C., to complete the reaction. After centrifuging, the supernatant was spectroscopically analysed for the liberation of uridine-5′-diphosphate, whereby a pyruvate-kinase coupling enzyme reaction was used. Preparations without hexose phosphate, as well as the measurement of the recovery of added uridine-5′-diphosphate act as controls.

EXAMPLES

Example 1

Clonina of genes of the sucrose-phosphate-synthase from potato

[0070] Poly-A+ RNA was isolated from large leaves of spinach plants as well as potato plants grown in the greenhouse. Resulting from the poly-A+ RNA, a CDNA library in the expression vector Lambda Zap II was laid out. 100,000 Plaques of both libraries were separated from spinach using a rabbit antiserum directed against pure SPS protein in relation to immunologically cross reacting protein. (Sonnewald et al., 1992, in press). From the potato library, positively reacting clones were obtained. These clones were further purified by standard methods and, by in vivo excision, plasmids were obtained which carried a double stranded cDNA as an insertion. After testing the size of the insertions, individual clones were analysed by determining the primary sequence.

Example 2

Determination of the Nucleotide Seauence of the SPS from Potato Codina cDNA Molecules and Derivation of the Corresponding Amino acid Sequences

[0071] The nucleotide sequences of the insertions obtained from Example 1, were determined by standard methods by means of the dideoxy method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467). The nucleotide sequences (Seq. ID No. 1 to Sea. ID No. 3) are described above. The amino acid sequences derived therefrom are also given.

Example 3

Construct of the Plasmid p35s-anti-pot-SPS and Insertion of Gene 35s-anti-pot-sps in the Genome of Potato Plants

[0072] The gene 35s-anti-pot-SPS consists of the three fragments A, B and C (see FIG. 1).

[0073] The plasmid was prepared as follows:

[0074] From the pbluescript plasmid with the total insertion, an approximately 2 kb size fragment was prepared by EcoRV cleavage, and this was cloned in the SmaI cleavage site of the vector pBinAR (Höfgen & Willmitzer, 1990, Plant Sci., 66-221-230). The vector pBinAR is a derivative of the binary vector BIN 19 (Bevan, 1984, Nucl. Acids Res. 12: 8711-8721-8721) and was transferred using an Agrobacterium tumefaciens mediated transformation into potato. Intact, fertile plants were regenerated from the transformed cells.

[0075] As a result of the transformation, some transgenic potato plants were shown to have a reduced amount of RNA coding for the potato SPS (see FIG. 3). 50 μg total RNA in a Northern blot experiment was hybridised with the probe for SPS from potato.

[0076] Further the plants showed a reduction in SPS activity (see Table I).

[0077] Thus, by the transfer and expression of the gene 35s-anti-pot-SPS in potato plants, the amount of mRNA for the SPS protein which is formed, as well as the existing enzymatic activity can be significantly reduced.

Example 4

Construct of Plasmid pB33-anti-pot-SpS and Insertion of Gene B33-anti-pot-SPS in the Genome of Potato Plants

[0078] The gene B33-anti-pot-SPS consists of the three fragments A, B and C (see FIG. 4). The plasmid was prepared in an analogous method to that described in Example 3, except a pBin 19 derivative was used as starting vector, that contains the B33 promoter of the patatin gene from Solanum tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8: 23-29) in place of the 35S promoter of pBinAR.

[0079] The gene B33-anti-pot-SPS was transferred using an Agrobacterium tumefaciens mediated transformation into potato. Intact, fertile plants were regenerated from the transformed cells.

[0080] As a result of the transformation, some transgenic potato plants were shown with a reduced amount of RNA coding for the potato SPS (see FIG. 4). 50 μg total RNA in a Northern blot experiment was hybridised with the probe for SPS from potato.

[0081] Further the plants also showed a reduction of the SPS activity only in the tubers.

[0082] Thus, by the transfer and expression of the gene 35s-anti-pot-SPS in potato plants, the amount of mRNA for the SPS protein which is formed, as well as the existing enzymatic activity can be significantly reduced.

2TABLE IResults of the transformation of potato plants12345Control26.13.613.81001-5511.82.722.9451-5720.45.928.9781-593.81.436.814.61-673.81.744.714.61-6917.22.011.7671-7214.61.913.0561-745.11.733.319.5Column 1: Control = Wild type plants, numbers indicate individual transgenic plants Column 2: Maximal speed of the enzyme reaction in the SPS activity test in nmol/min/mg. Column 3: Speed in the SPS activity test in nmol/min/mg. Column 4: Activity level of the SPS in %. Column 5: Residual activity of the SPS in %.

	Number	Date	Country
Parent	08778656	Jan 1997	US
Child	09376045	Aug 1999	US

DNA SEQUENCES AND PLASMIDS FOR THE PREPARATION OF PLANTS WITH CHANGED SUCROSE CONCENTRATION

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