STARCH BRANCHING ENZYME II OF POTATO

Abstract: The present invention relates to an amino acid sequence of second starch branching enzyme (SBE II) of potato and a fragment thereof as well as to the corresponding isolated DNA sequences. Furthermore, the invention relates to vectors comprising such an isolated DNA sequence, to processes for production of transgenic potatoes, and to the use of said potatoes for the production of starch. The starch obtained will show a changed pattern of branching of amylopectin as well as a changed amylose/amylopectin ratio.

Description: STARCH BRANCHING ENZYME II OF POTATO The present invention relates to a novel starch branching enzyme of potato. More specifically, the present invention relates to an amino acid sequence of a second starch branching enzyme (SBE II) of potato and a fragment thereof as well as their corresponding DNA sequences. Furthermore, the invention relates to vectors comprising such DNA sequences, to processes for production of transgenic potatoes, and to the use of said potatoes for the production of starch. Starch is a complex mixture of different molecule forms differing in degree of polymerization and branching of the glucose chains. Starch consists of amylose and amylopectm, whereby the amylose consists of an essentially linear α-l,4-glucan and amylopectin consists of α-1, 4-glucans connected to each other via α-1, 6- lmkages and, thus, forming a branched polyglucan. Thus, starch is not a uniform raw material. Starch is synthesized via at least three enzymatic reactions in which ADP glucose phosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21) and starch branching enzyme (EC 2.4.1.18) are involved. Starch branching enzyme (SBE, also called Q-enzyme) is believed to have two different enzymatic activities. It catalyzes both the hydrolysis of α-1, -glucosιdιc bonds and the formation of α-1, 6-glucosιdιc bonds during synthesis of the branched component m starch, i.e. amylopectin. Plant starch is a valuable source of renewable raw material used in, for example, the chemical industry (Visser and Jacobsen, 1993) . However, the quality of the starch has to meet the demands of the processing industry wherein uniformity of structure is an important criterion. For industrial application there is a need of plants only containing amylose starch and plants only containing amylopectin starch, respectively. Processes for altering the a ylose/amylooectin ratio in starch have already been proposed. For exanple, m WO95/04826 there is described DNA sequences encoding debranching enzymes with the ability to reduce or increase the degree of branching of amylopectin in transgenic plants, e.g. potatoes. In 092/14827 plasmids are described having DNA sequences that after insertion into the genome of the plants cause changes in the carbohydrate concentration and the carbohydrate composition in regenerated plants. These changes can be obtained from a sequence of a oranchmg enzyme that is located on these plasmids. This branching enzyme is proposed to alter the amylose/amylopectin ratio m starch of the plants, especially in commercially used plants. 092/14827 describes the only hitherto known starch branching enzyme potato and within the art it is not known whether other starch branching enzymes are involved in the synthesis of branched starch of potato. In Mol Gen Genet (1991) 225:289-296, Visser et al., there is described inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Inhibition of the enzyme m potato tuber starch was up to 100% in which case amylose-free starch was provided. However, the prior known methods for inhibiting amylopectin have not been that successful anc, therefore, alternative methods for inhibiting amylopectin are still highly desirable (Muller-Rober and Koβmann, 1994; Martin and Smith, 1995) . The object of the present invention is to enable altering the degree of amylopectm branching and the amylopectin/amylose ratio in potato starch. According to the present invention this object is achieved by providing a novel isolated DNA sequence encoding a second starch branching enzyme, SBE II, and fragments thereof, which after insertion into the genome of the plants cause changes said branching degree and ratio m regenerated plants. With the scope of the present invention there is also included the ammo acid sequence of SBE II and fragments thereof. Also variants of the above DNA sequence resulting from the degeneracy of the genetic code are encompassed. The novel DNA sequence encoding SBEII, comprising 3074 nucleotides, as well as the corresponding am o acid sequence comprising 878 ammo acids, are shown in SEQ ID No. 1. One 1393 nucleotides long fragment of the above DNA sequence, correspondmg to nucleotides 1007 to 2399 of the DNA sequence in SEQ ID No. 1, as well as the correspondmg ammo acid sequence comprising 464 ammo acids, are shown m SEQ ID No. 2. Furthermore, there are provided vectors comprising said isolated DNA-sequences and regulatory elements active in potato. The DNA sequences may be inserted in the sense or antisense (reversed) orientation in the vectors in relation to a promoter immediately upstream from the DNA sequence. Also there is provided a process for the production of transgenic potatoes with a reduced degree of branching of amylopect starch, comprising the following steps: a) transfer and incorporation of a vector according to the invention into the genome of a potato cell, and b) regeneration of intact, whole plants from the transformed cells. Finally, the invention provides the use of said transgenic potatoes for the production of starch. The invention will be described in more detail below in association with an experimental part and the accompanying drawings, in which Fig. 1 shows SDS polyacrylamide electrophoresis of proteins extracted from starch of normal potato (lane A) and transgenic potato (lane B) . Excised protein bands are marked with arrows. Lane M: Molecular weight marker proteins (kDa) . Fig. 2 shows 4 peptide sequences derived from digested proteins from potato tuber starch. EXPERIMENTAL PART Isola tion of s tarch from pota to tubers Potato plants { Solanum tuberosum) were grown in the field. Peeled tubers from either cv. Early Puritan or from a transgenic potato line essentially lacking granule-bound starch synthase I (Svalof Weibull AB, international application number PCT/SE91/00892) , were homogenized at 4°C in a fruit juicer. To the "juice fraction", which contained a large fraction of the starch, was immediately added Tris-HCl, pH 7.5, to 50 mM, Na-dithiomte to 30 mM and ethylenedimtrilotetraacetic acid (EDTA) to 10 mM. The starch granules were allowed to sediment for 20 mm and washed 4x with 10 bed volumes of washing buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA) . The starch, which was left on the bench at +4°C for 30 mm to sediment between every wash, was finally washed with 3 3 bed volumes of acetone, air dried over night, and stored at -20°C. Extraction of proteins from tuber starch Stored starch (20 g) was continuously mixed with 200 ml extraction buffer (50 mM Tris-HCl, pH 7.5, 2% (w/v) sodium dodecyl sulfate (SDS) , 5 mM EDTA) by aspiration with a pipette at 85°C until the starch was gelatinized. The samples were then frozen at -70°C for 1 hour. After thawing at 50°C, the samples were centrifuged for 20 mm at 12,000xg at 10°C. The supernatants were coLlected and re-centπfuged at 3,000xg for 15 mm. The final superna¬ tants were filtered through 0.45 μ filters and 2.25 volumes of ice-cold acetone were added. After 30 mm incubation at 4°C, the protein precipitates were collected by centrifugation (3,000xg for 30 mm at 4°C) , and dissolved in 50 mM Tris-HCl, pH 7.5. An aliquot of each preparation was analyzed by SDS poly-acrylamide gel electrophoresis according to Laemmli (1970) (Fig. 1) . The proteins in the remaining portions of the preparations were concentrated by precipitation with trichloroacetic acid (10%) and the proteins were separated on an 8% SDS polyacrylamide gel Laemmli, (1970) . The proteins the gel were stained with Coomassie Brilliant Blue R-250 (0.2% in 20% methanol, 0.5% acetic acid, 79.5% H 2 0) . In gel digestion and sequencing of peptides The stained bands marked with arrows Fig. 1 corresponding to an apparent molecular weight of about 100 kDa were excised and washed twice with 0.2M NH 4 HCO in 50° acetonitrile under continuous stirring at 35°C for 20 mm. After each washing, the liquid was removed and the gel pieces were allowed to dry by evaporation in a fume hood. The completely dried gel pieces were then separately placed on parafilm and 2 μl of 0.2M NH 4 C0 3 , 0.02% Tween-20 were added. Modified trypsin (Promega, Madison, I,UΞA) (0.25 μg in 2 μl) was sucked into the gel pieces whereafter 0.2M NH 4 C0 3 was added in 5 μl portions until they had resumed their original sizes. The gel slices were further divided mto three pieces and transferred to an Eppendorf tube. 0.2M NH 4 C0 3 (200 μl) was added and the proteins contained m the gel pieces were digested over night at 37°C (Rosenfeld et al. 1992) . After completed digestion, trifluoroacetic acid was added to 1% and the supernatants removed and saved. The gel pieces were further extracted twice with 60% acetonitrile, 0.1% tπ- fluoroacetic acid (200 μl) under continuous shaking at 37°C for 20 mm. The two supernatants from these extrac¬ tions were combined with the first supernatant. The gel pieces were finally washed with 60% acetonitrile, 0.11 trifluoroacetic acid, 0.02% Tween-20 (200 μl) . Also these supernatants were combined with the other supernatants and the volume was reduced to 50 μl by evaporation. The extracted peptides were separated on a SMART ® chromato¬ graphy system (Pharmacia, Uppsala, Sweden) equipped with a μRPC C2/C18 SC2.1/10 column. Peptides were elited with a gradient of 0 - 60% acetonitrile in water/0.11 trifluoro- acetic acid over 60 min with a flow rate of ICO μl/mm. Peptides were sequenced either on an Applied Biosystems 470A gas phase sequenator with an on line PTH- ammo acid analyzer (120A) or on a model 476A according to the instructions of the manufacturer (Applied Biosystems, Foster City, CA, USA) . Four of the peptides sequenced gave easiJy terpretable sequences (Fig. 2) . A data base search revealed that these four peptides displayed similarity to starch branching enzymes and interestingly, the peptides were more related to starch branching enzyme ! " I from other plant species than to starch branching enzyme 1 from potato. Construction of oligonucleotides encoding pep '.ides 1 and 2. Degenerated oligonucleotides encodmg peptide 1 and peptide 2 were synthesized as forward and reverse primers, respectively: Oligonucleotide 1: 5' -gtaaaacgacggccagt- TTYGGNGTNTGGGARATHTT-3' (Residues 2 to 8 of peptide 1) Oligonucleotide 2: 5 ' -aattaaccctcactaaaggg- CKRTCRAAYTCYTGIARNCC-3' (Residues 2 to 8 of psptide 2, reversed strand) wherein H is A, C or T, I is mosine; K is G or T; N is A, C, G or T; R is A or G; Y is C or T; bases in lower case were added as tag sequences. Purifi ca tion of mRNA from pota to tuber, syn thesis of cDNA and PCR amplifica tion of a cDNA fragment corresponding to pota to starch branching enzyme II . Total RNA from mature potato tubers ( S . tuberosum cv. Amanda) was isolated as described (Logemann et al. 1987) . First strand cDNA was synthesized using 2 μg of total RNA and 60 pmol of olιgo-dT 30 as downstream primer. The primer was annealed to the polyA of the mRNA at 60°C for 5 mm. The extension of the cDNA was performed according to the technical manual of the manufacturer using the Rιboclone c cDNA Synthesis System M-MLV (H-) (Promega) . cDNA encodmg the novel starch branching enzyme II according to the invention was amplified in a Perkm-Elmer GeneAmp® 9600 PCR ther ocycler (Perkm-Elmer Cetus Instruments, CT, USA) using the two degenerate primers designed from the peptides 1 and 2 (see above) under the following conditions: 1 mM dNTP, 1 μM of each primer and an alicot of the cDNA described above in a total reaction volume of 20 μl with lx AmpliTaq® buffer and 0,8 U AmpliTaq® (Perkm-Elmer Cetus) . The cycling conditions were: 96°C for 1', 80°C while the enzyme was added as a hotstart (approximately 15'), an unintended drop to 25°C, five cycles of 94°C for 20", 45°C for 1', ramp to 72°C for 1' and 72°C for 2', and 30 cycles of 94°C for 5", 45°C for 30", and 72°C for (2'+2" per cycle) and completed with 72°C for 10' prior to chilling to 4°C. A sample of this reaction (0.1 μl) was reamplified usmg the cycling conditions: 96°C for 1', 80°C while the enzyme was added as a hotstart (approximately 5'), five cycles of 94°C for 20' ', 45°C for 1', and 72°C for 2', and 25 cycles of 94°C for 5' ', 45°C for 30' ', and 72°C for (2' + 2' ' per cycle) and completed with 72°C for 10' prior to chilling to 4°C. After completion of the PCR ampli¬ fication, the reaction was loaded on a 1.5% Seakem agarose gel (FMC Bioproducts, Rockland, ME, USA) . After electrophoresis and staining with ethidium bromide a major band with an apparent size of 1500 bp was exc sed and the fragment was eluted by shaking m water (200 μl) for 1 h. This fragment was used as template in sequenc ng reactions after reamplification using primers corresponding to the tag sequences (in oligonucleotides 1 and 2) , purification by agarose gel electrophoresis as above and extraction from the gel using the Qiaex ® gel extraction kit accordmg to the manufacturer s instructions (DIAGEN GmbH, Hilden, Germany) . The sequencing reactions were done using the DyeDeoxy® Terminator Cycle Sequencing kits (Perkm-Elmer Cetus Instruments) using tag sequences and inl ernal primers. The sequencing reaction were analyzed on an Applied Biosystems 373A DNA sequencer according to the manufacturer s protocols. The sequence was ed ted and comprised 1393 bp. To complete the determination of the sequence of starch branching enzyme II, the 5' and 3' ends of the full length cDNA were amplified from the same tota RNA as above using rapid amplification of cDNA ends, RACE, methodology with specific primers from the 1393 bp sequence. In the 3' end amplification, an oligo T^G primer was used against the poly A tail and in the 5 end, the 5'/3' RACE kit from Boehrmger Mannheim (Cat. No. 1734792) was used. The fragments from these amplifications were sequenced in the same way as above usmg te 'nal and end primers. The sequences from the two ends were aligned together with the 1393 base pairs to give a composite full length cDNA sequence. Primers were designed from this sequence to amplify the whole coding region n one part. Partial sequencing of the amplified coding cDxlA confirmed the presence of a cDNA correspondmg to the composite sequence. The full length cDNA is 3074 bp and the translated sequence comprises 878 ammo acids. The mature protein comprises 830 ammo acids. Comparisons of the consensus sequence with the EMBL and GenBank databases showed 68% identity to ootato starch branching enzyme I and about 80% identity to starch branching enzyme II from other plant species. The present inventors therefore denote the enzyme encoded by the new branching enzyme sequence potato starch branching enzyme II. Transforma tion of pota to plants The isolated full length cDNA of potato starch branching enzyme II and other functionally active frag¬ ments the range of 50-3 074 bp are cloned reverse orientation behind promoters active in potato tubers. By the term "functionally active" is meant fragments that will affect the amylose/amylopectin ratio in potato starch. The DNA and ammo acid sequence of SBE II accordmg to the mvention as well as one fragment of the DNA and correspondmg ammo acid sequence are shown in SEQ ID No. 1 and 2, respectively. The promoters are selected from, for example, the patatm promoter, the promoter from the potato granule- bound starch synthase I gene or promoters isolated from potato starch branching enzymes I and II genes. The constructs are cloned by techniques known in the art either a binary Ti-plasmid vector suitable for transformation of potato mediated by Agrobacterium tumefa ciens, or in a vector suitable for direct transformation using ballistic techniques or electroporation. It is realized that the sense (see below) and antisense constructs must contain all necessary regulatory elements. Transgenic potato plants transcribe the inverse starch branching enzyme II construct specifically in tubers, leading to antisense inhibition of the enzyme. A reduction and changed pattern of the branching of amylo¬ pect as well as a changed amylose/amylopectin ratio thereby occur tuber starch. The antisense construct for potato starch branching enzyme II is also used in combination with antisense 10 constructs for potato starch branching enzyme I, for potato granule-bound starch synthase II, for potato soluble starch synthases II and III, for potato starch disproportionating enzyme (D-enzyme) or for potato starch debranching enzyme to transform potato to change the degree of branching of amylopectin and the amylose/amylopectin ratio. This gives new and valuable raw material to the starch processing industry. The full-length cDNA sequence encoding the enzyme is, in different constructs, cloned m sense orientation behind one or more of the promoters mentioned above, and the constructs are transferred into suitable transforma¬ tion vectors as described above and used for the transformation of potato. Regenerated transformed potato plants will produce an excess of starch branching enzyme II in the tubers leading to an increased degree and changed pattern of branching of amylopectin cr to inhibition of transcription of endogenous starch branching enzyme II transcription due to co-suppression, resulting in a decreased branching of .amylopectm. References Muller-Rober, B., Koβmann, J. , (1994) Approaches to influence starch quantity and starch quality in transgenic plants. Plant Cell Env ronm. 17, 601-613. Martin, C, Smith, A. (1995) Starch Biosynthesis. Plant Cell 7, 971-985. Laemmli, U.K. (1979) Cleavage of structural proteins during assembly of the head of bacteriophage T . Nature 227, 680-685. Logemann, J., Schell, J. and Willmitzer, L. (1987) Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163, 16-20. Rosenfeld, J. , Capdeville, J, Guillemot, J.C, Ferrara, P. (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173-179. Visser, R.G.F., Jacobsen, E. (1993) Towards modifying plants for altered starch content and composition. TibTech 11, 63-68. SEQ ID No . 1 Sequenced molecule: cDNA Name: bell gene (branching enzyme II) from Solanum tuberosum (potato) Length of sequence: 3074 bp AAACCTCCTC CACTCAGTCT TTGTTTCTCT CTCTCTTCAC GCTTCTCTTG GCGCCTTGJA 60 CTCAGCAATT TGACACTCAG TTAGTTACAC TNCCATCACT TATCAGATCT CTATTTTTVC 120 TCTTAATTCC AACCAAGGAA TGAATAAAAA GATAGATTTG TAAAAACCCT AAGGAGAGi-A 180 GAAGAAAG ATG GTG TAT ACA CTC TCT GGA GTT CGT TTT CCT ACT GTT CCΛ 230 Met Val Tyr Thr Leu Ser Gly Val Arg Phe Pro Thr Val Pro -45 -40 -3.'j TCA GTG TAC AAA TCT AAT GGA TTC AGC AGT AAT GGT GAT CGG AGG AAT 278 Ser Val Tyr Lys Ser Asn Gly Phe Ser Ser Asn Gly Asp Arg Arg Asn -30 -25 -20 GCT AAT NTT TCT GTA TTC TTG AAA AAG CAC TCT CTT TCA CGG AAG ATC 326 Ala Asn Xaa Ser Val Phe Leu Lys Lys His Ser Leu Ser Arg Lys He -15 -10 -5 TTG GCT GAA AAG TCT TCT TAC AAT TCC GAA TCC CGA CCT TCT ACA GTT 374 Leu Ala Glu Lys Ser Ser Tyr Asn Ser Glu Ser Arg Pro Ser Thr Val 1 5 10 GCA GCA TCG GGG AAA GTC CTT GTG CCT GGA ACC CAG AGT GAT AGC TCC 422 Ala Ala Ser Gly Lys Val Leu Val Pro Gly Thr Gin Ser Asp Ser Ser 15 20 25 30 TCA TCC TCA ACA GAC CAA TTT GAG TTC ACT GAG ACA TCT CCA GAA AAT 470 Ser Ser Ser Thr Asp Gin Phe Glu Phe Thr Glu Thr Ser Pro Glu Asn 35 40 45 TCC CCA GCA TCA ACT GAT GTA GAT AGT TCA ACA ATG GAA CAC GCT AGC 518 Ser Pro Ala Ser Thr Asp Val Asp Ser Ser Thr Met Glu His Ala Ser 50 55 60 CAG ATT AAA ACT GAG AAC GAT GAC GTT GAG CCG TCA AGT GAT CTT ACS 566 Gin He Lys Thr Glu Asn Asp Asp Val Glu Pro Ser Ser Asp Leu Thr 65 70 75 GGA AGT GTT GAA GAG CTG GAT TTT GCT TCA TCA CTA CAA CTA CAA GA? 614 Gly Ser Val Glu Glu Leu Asp Phe Ala Ser Ser Leu Gin Leu Gin Glv 80 85 90 GGT GGT AAA CTG GAG GAG TCT AAA ACA TTA AAT ACT TCT GAA GAG AC/ 662 Gly Gly Lys Leu Glu Glu Ser Lys Thr Leu Asn Thr Ser Glu Glu Thi 95 100 105 IIC ATT ATT GAT GAA TCT GAT AGG ATC AGA GAG AGG GGC ATC CCT CCA CCT 710 He He Asp Glu Ser Asp Arg He Arg Glu Arg Gly He Pro Pro Pro 115 120 125 GGA CTT GGT CAG AAG ATT TAT GAA ATA GAC CCC CTT TTG ACA AAC TA7 758 Gly Leu Gly Gin Lys He Tyr Glu He Asp Pro Leu Leu Thr Asn Tyr 130 135 140 CGT CAA CAC CTT GAT TAC AGG TAT TCA CAG TAC AAG AAA CTG AGG GAG 806 Arg Gin His Leu Asp Tyr Arg Tyr Ser Gin Tyr Lys Lys Leu Arg Glu 145 150 155 GCA ATT GAC AAG TAT GAG GGT GGT TTG GAA GCT TTT TCT CGT GGT TAT 854 Ala He Asp Lys Tyr Glu Gly Gly Leu Glu Ala Phe Ser Arg Gly Tyr 160 165 170 GAA AAA ATG GGT TTC ACT CGT AGT GCT ACA GGT ATC ACT TAC CGT GAG 902 Glu Lys Met Gly Phe Thr Arg Ser Ala Thr Gly He Thr Tyr Arg Glu 175 180 185 190 TGG GCT CCT GGT GCC CAG TCA GCT GCC CTC ATT GGA GAT TTC AAC AAT 950 Trp Ala Pro Gly Ala Gin Ser Ala Ala Leu He Gly Asp Phe Asn Asn 195 200 205 TGG GAC GCA AAT GCT GAC ATT ATG ACT CGG AAT GAA TTT GGT GTC TGG 998 Trp Asp Ala Asn Ala Asp He Met Thr Arg Asn Glu Phe Gly Val Trp 210 215 220 GAG ATT TTT CTG CCA AAT AAT GTG GAT GGT TCT CCT GCA ATT CCT CAT 1046 Glu He Phe Leu Pro Asn Asn Val Asp Gly Ser Pro Ala He Pro His 225 230 235 GGG TCC AGA GTG AAG ATA CGT ATG GAC ACT CCA TCA GGT GTT AAG GAT 1094 Gly Ser Arg Val Lys He Arg Met Asp Thr Pro Ser Gly Val Lys Asp 240 245 250 TCC ATT CCT GCT TGG ATC AAC TAC TCT TTA CAG CTT CCT GAT GAA ATT 1142 Ser He Pro Ala Trp He Asn Tyr Ser Leu Gin Leu Pro Asp Glu He 255 260 265 270 CCA TAT AAT GGA ATA TAT TAT GAT CCA CCC GAA GAG GAG AGG TAT ATC 1190 Pro Tyr Asn Gly He Tyr Tyr Asp Pro Pro Glu Glu Glu Arg Tyr He 275 280 285 TTC CAA CAC CCA CGG CCA AAG AAA CCA AAG TCG CTG AGA ATA TAT GAA 1238 Phe Gin His Pro Arg Pro Lys Lys Pro Lys Ser Leu Arg He Tyr Glu 290 295 300 TCT CAT ATT GGA ATG AGT AGT CCG GAG CCT AAA ATT AAC TCA TAC GTG 1286 Ser His He Gly Met Ser Ser Pro Glu Pro Lys He Asn Ser Tyr Val 305 310 315 AAT TTT AGA GAT GAA GTT CTT CCT CGC ATA AAA AAG CTT GGG TAC AAT 1334 Asn Phe Arg Asp Glu Val Leu Pro Arg He Lys Lys Leu Gly Tyr Asn 320 325 330 GCG GTG CAA ATT ATG GCT ATT CAA GAG CAT TCT TAT TAT GCT AGT TTT 1382 Ala Val Gin He Met Ala He Gin Glu His Ser Tyr Tyr Ala Ser Phe 335 340 345 350 GGT TAT CAT GTC ACA AAT TTT TTN GCA CCA AGC AGC CGT TTT GGA ACN 1430 Gly Tyr His Val Thr Asn Phe Xaa Ala Pro Ser Ser Arg Phe Gly Thr 355 360 365 CCC GAC GAC CTT AAG TCT TTG ATT GAT AAA GCT CAT GAG CTA GGA ATT 1478 Pro Asp Asp Leu Lys Ser Leu He Asp Lys Ala His Glu Leu Gly He 370 375 380 GTT GTT CTC ATG GAC ATT GTT CAC AGC CAT GCA TCA AAT AAT ACT TTA 1526 Val Val Leu Met Asp He Val His Ser His Ala Ser Asn Asn Thr Leu 385 390 395 GAT GGA CTG AAC ATG TTT GAC GGC ACA GAT AGT TGT TAC TTT CAC TCT 1574 Asp Gly Leu Asn Met Phe Asp Gly Thr Asp Ser Cys Tyr Phe His Ser 400 405 410 GGA GCT CGT GGT TAT CAT TGG ATG TGG GAT TCC CGC CTC TTT AAC TAT 1622 Gly Ala Arg Gly Tyr His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr 415 420 425 430 GGA AAC TGG GAG GTA CTT AGG TAT CTT CTC TCA AAT GCG AGA TGG TGG 1670 Gly Asn Trp Glu Val Leu Arg Tyr Leu Leu Ser Asn Ala Arg Trp Trp 435 440 445 TTG GAT GAG TTC AAA TTT GAT GGA TTT AGA TTT GAT GGT GTG ACA TCA 1718 Leu Asp Glu Phe Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser 450 455 460 ATG ATG TAT ACT CAC CAC GGA TTA TCG GTG GGA TTC ACT GGG AAC TAC 1766 Met Met Tyr Thr His His Gly Leu Ser Val Gly Phe Thr Gly Asn Tyr 465 470 475 GAG GAA TAC TTT GGA CTC GCA ACT GAT GTG GAT GCT GTT GTG TAT CTG 1814 Glu Glu Tyr Phe Gly Leu Ala Thr Asp Val Asp Ala Val Val Tyr Leu 480 485 490 ATG CTG GTC AAC GAT CTT ATT CAT GGG CTT TTC CCA GAT GCA ATT ACC 1862 Met Leu Val Asn Asp Leu He His Gly Leu Phe Pro Asp Ala He Thr 495 500 505 510 ATT GGT GAA GAT GTT AGC GGA ATG CCG ACA TTT TNT ATT CCC GTT CAA 1910 He Gly Glu Asp Val Ser Gly Met Pro Thr Phe Xaa He Pro Val Gin 515 520 525 GAT GGG GGT GTT GGC TTT GAC TAT CGG CTG CAT ATG GCA ATT GCT GAT 1958 Asp Gly Gly Val Gly Phe Asp Tyr Arg Leu His Met Ala He Ala Asp 530 535 540 AAA TGG ATT GAG TTG CTC AAG AAA CGG GAT GAG GAT TGG AGA GTG GGT 2006 Lys Trp He Glu Leu Leu Lys Lys Arg Asp Glu Asp Trp Arg Val Gly 545 550 555 GAT ATT GTT CAT ACA CTG ACA AAT AGA AGA TGG TCG GAA AAG TGT GTT 2054 Asp He Val His Thr Leu Thr Asn Arg Arg Trp Ser Glu Lys Cys Val 560 565 570 TCA TAC GCT GAA AGT CAT GAT CAA GCT CTA GTC GGT GAT AAA ACT ATA 2102 Ser Tyr Ala Glu Ser His Asp Gin Ala Leu Val Gly Asp Lys Thr He 575 580 585 590 GCA TTC TGG CTG ATG GAC AAG GAT ATG TAT GAT TTT ATG GCT CTG GAT 2150 Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp 595 600 605 AGA CCN TCA ACA TCA TTA ATA GAT CGT GGG ATA GCA TTG CAC AAG ATG 2198 Arg Pro Ser Thr Ser Leu He Asp Arg Gly He Ala Leu His Lys Met 610 615 620 ATT AGG CTT GTA ACT ATG GGA TTA GGA GGA GAA GGG TAC CTA AAT TTC 2246 He Arg Leu Val Thr Met Gly Leu Gly Gly Glu Gly Tyr Leu Asn Phe 625 630 635 ATG GGA AAT GAA TTC GGC CAC CCT GAG TGG ATT GAT TTC CCT AGG GCT 2294 Met Gly Asn Glu Phe Gly His Pro Glu Trp He Asp Phe Pro Arg Ala 640 645 650 GAA CAA CAC CTC TCT GAT GGC TCA GTA ATT CCC GGA AAC CAA TTC AGT 2342 Glu Gin His Leu Ser Asp Gly Ser Val He Pro Gly Asn Gin Phe Ser 655 660 665 670 TAT GAT AAA TGC AGA CGG AGA TTT GAC CTG GGA GAT GCA GAA TAT TTA 2390 Tyr Asp Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala Glu Tyr Leu 675 680 685 AGA TAC CGT GGG TTG CAA GAA TTT GAC CGG GCT ATG CAG TAT CTT GAA 2438 Arg Tyr Arg Gly Leu Gin Glu Phe Asp Arg Ala Met Gin Tyr Leu Glu 690 695 700 GAT AAA TAT GAG TTT ATG ACT TCA GAA CAC CAG TTC ATA TCA CGA AAG 2486 Asp Lys Tyr Glu Phe Met Thr Ser Glu His Gin Phe He Ser Arg Lys 705 710 715 GAT GAA GGA GAT AGG ATG ATT GTA TTT GAA AAA GGA AAC CTA GTT TTT 2534 Asp Glu Gly Asp Arg Met He Val Phe Glu Lys Gly Asn Leu Val Phe 720 725 730 GTC TTT AAT TTT CAC TGG ACA AAA AGC TAT TCA GAC TAT CGC ATA GGC 2582 Val Phe Asn Phe His Trp Thr Lys Ser Tyr Ser Asp Tyr Arg He Gly 735 740 745 750 TGC CTG AAG CCT GGA AAA TAC AAG GTT GCC TTG GAC TCA GAT GAT CCA 2630 Cys Leu Lys Pro Gly Lys Tyr Lys Val Ala Leu Asp Ser Asp Asp Pro 755 760 765 CTT TTT GGT GGC TTC GGG AGA ATT GAT CAT AAT GCC GAA TAT TTC ACC 2678 Leu Phe Gly Gly Phe Gly Arg He Asp His Asn Ala Glu Tyr Phe Thr 770 775 780 TTT GAA GGA TGG TAT GAT GAT CGT CCT CGT TCA ATT ATG GTG TAT GCA 2721 Phe Glu Gly Trp Tyr Asp Asp Arg Pro Arg Ser He Met Val Tyr Ala 785 790 795 CCT AGT AGA ACA GCA GTG GTC TAT GCA CTA GTA GAC AAA GAA GAA GAA 2774 Pro Ser Arg Thr Ala Val Val Tyr Ala Leu Val Asp Lys Glu Glu Glu 800 805 810 GAA GAA GAA GAA GTA GCA GTA GTA GAA GAA GTA GTA GTA GAA GAA GAA 2822 Glu Glu Glu Glu Val Ala Val Val Glu Glu Val Val Val Glu Glu Glu 815 820 825 830 TGA ACGAA CTTGTGATCG CGTTGAAAGA TTTGAAGGCT ACATAGAGCT TCTTGACGTA 2880 *** TCTGGCAATA TTGCATCAGT CTTGGCGGAA TTTCATGTGA CAAAAGGTTT GCAATTCTTT 29 0 CCACTATTAG TAGTGCAACG ATATACGCAG AGATGAAGTG CTGCACAAAC ATATGTAAAA 3000 TCGATGAATT TATGTCGAAT GCTGGGACGG GCTTCAGCAG GTTTTGCTTA GTGAGTTCTG 3060 TAAATTGTCA TCTC 3074 SEQ ID No . 2 Sequenced molecule: cDNA Name: bell gene fragment (branching enzyme II) from Solanum tuberosum (potato) Length of sequence: 1393 bp T CTG CCA AAT AAT GTG GAT GGT TCT CCT GCA ATT CCT CAT GGG TCC A'3A 49 Leu Pro Asn Asn Val Asp Gly Ser Pro Ala He Pro His Gly Ser Arg 1 5 10 15 GTG AAG ATA CGT ATG GAC ACT CCA TCA GGT GTT AAG GAT TCC ATT CCT 97 Val Lys He Arg Met Asp Thr Pro Ser Gly Val Lys Asp Ser He Pro 20 25 30 GCT TGG ATC AAC TAC TCT TTA CAG CTT CCT GAT GAA ATT CCA TAT AAT 145 Ala Trp He Asn Tyr Ser Leu Gin Leu Pro Asp Glu He Pro Tyr Asn 35 40 45 GGA ATA TAT TAT GAT CCA CCC GAA GAG GAG AGG TAT ATC TTC CAA CAC 193 Gly He Tyr Tyr Asp Pro Pro Glu Glu Glu Arg Tyr He Phe Gin His 50 55 60 CCA CGG CCA AAG AAA CCA AAG TCG CTG AGA ATA TAT GAA TCT CAT ATT 241 Pro Arg Pro Lys Lys Pro Lys Ser Leu Arg He Tyr Glu Ser His He 65 70 75 80 GGA ATG AGT AGT CCG GAG CCT AAA ATT AAC TCA TAC GTG AAT TTT AGA 289 Gly Met Ser Ser Pro Glu Pro Lys He Asn Ser Tyr Val Asn Phe Arg 85 90 95 GAT GAA GTT CTT CCT CGC ATA AAA AAG CTT GGG TAC AAT GCG GTG CA? 337 Asp Glu Val Leu Pro Arg He Lys Lys Leu Gly Tyr Asn Ala Val Glr 100 105 110 ATT ATG GCT ATT CAA GAG CAT TCT TAT TAT GCT AGT TTT GGT TAT CAT 385 He Met Ala He Gin Glu His Ser Tyr Tyr Ala Ser Phe Gly Tyr His 115 120 125 GTC ACA AAT TTT TTN GCA CCA AGC AGC CGT TTT GGA ACN CCC GAC GAC 433 Val Thr Asn Phe Xaa Ala Pro Ser Ser Arg Phe Gly Thr Pro Asp Asp 130 135 140 CTT AAG TCT TTG ATT GAT AAA GCT CAT GAG CTA GGA ATT GTT GTT CTC 481 Leu Lys Ser Leu He Asp Lys Ala His Glu Leu Gly He Val Val Leu 145 150 155 160 ATG GAC ATT GTT CAC AGC CAT GCA TCA AAT AAT ACT TTA GAT GGA CTG 529 Met Asp He Val His Ser His Ala Ser Asn Asn Thr Leu Asp Gly Leu 165 170 175 AAC ATG TTT GAC GGC ACA GAT AGT TGT TAC TTT CAC TCT GGA GCT CGT 577 Asn Met Phe Asp Gly Thr Asp Ser Cys Tyr Phe His Ser Gly Ala Arg 180 185 190 GGT TAT CAT TGG ATG TGG GAT TCC CGC CTC TTT AAC TAT GGA AAC TGΞ 625 Gly Tyr His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Asn Trs 195 200 205 GAG GTA CTT AGG TAT CTT CTC TCA AAT GCG AGA TGG TGG TTG GAT GA3 673 Glu Val Leu Arg Tyr Leu Leu Ser Asn Ala Arg Trp Trp Leu Asp Giα 210 215 220 TTC AAA TTT GAT GGA TTT AGA TTT GAT GGT GTG ACA TCA ATG ATG TAT 721 Phe Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met Tyr 225 230 235 240 ACT CAC CAC GGA TTA TCG GTG GGA TTC ACT GGG AAC TAC GAG GAA TAC 769 Thr His His Gly Leu Ser Val Gly Phe Thr Gly Asn Tyr Glu Glu Tyr 245 250 255 TTT GGA CTC GCA ACT GAT GTG GAT GCT GTT GTG TAT CTG ATG CTG GTC 812 Phe Gly Leu Ala Thr Asp Val Asp Ala Val Val Tyr Leu Met Leu Val 260 265 270 AAC GAT CTT ATT CAT GGG CTT TTC CCA GAT GCA ATT ACC ATT GGT GAA 865 Asn Asp Leu He His Gly Leu Phe Pro Asp Ala He Thr He Gly Glu 275 280 285 GAT GTT AGC GGA ATG CCG ACA TTT TNT ATT CCC GTT CAA GAT GGG GGT 913 Asp Val Ser Gly Met Pro Thr Phe Xaa He Pro Val Gin Asp Gly Gly 290 295 300 GTT GGC TTT GAC TAT CGG CTG CAT ATG GCA ATT GCT GAT AAA TGG ATT 961 Val Gly Phe Asp Tyr Arg Leu His Met Ala He Ala Asp Lys Trp He 305 310 315 320 GAG TTG CTC AAG AAA CGG GAT GAG GAT TGG AGA GTG GGT GAT ATT GTT 1019 Glu Leu Leu Lys Lys Arg Asp Glu Asp Trp Arg Val Gly Asp He Val 325 330 335 CAT ACA CTG ACA AAT AGA AGA TGG TCG GAA AAG TGT GTT TCA TAC GCT 1057 His Thr Leu Thr Asn Arg Arg Trp Ser Glu Lys Cys Val Ser Tyr Ala 340 345 350 GAA AGT CAT GAT CAA GCT CTA GTC GGT GAT AAA ACT ATA GCA TTC TGG 1105 Glu Ser His Asp Gin Ala Leu Val Gly Asp Lys Thr He Ala Phe Trp 355 360 365 CTG ATG GAC AAG GAT ATG TAT GAT TTT ATG GCT CTG GAT AGA CCN TCA 1153 Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro Ser 370 375 380 ACA TCA TTA ATA GAT CGT GGG ATA GCA TTG CAC AAG ATG ATT AGG CTT 1201 Thr Ser Leu He Asp Arg Gly He Ala Leu His Lys Met He Arg Leu 385 390 395 400 GTA ACT ATG GGA TTA GGA GGA GAA GGG TAC CTA AAT TTC ATG GGA AAT 1249 Val Thr Met Gly Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly Asn 405 410 415 GAA TTC GGC CAC CCT GAG TGG ATT GAT TTC CCT AGG GCT GAA CAA CAC 1297 Glu Phe Gly His Pro Glu Trp He Asp Phe Pro Arg Ala Glu Gin His 420 425 430 CTC TCT GAT GGC TCA GTA ATT CCC GGA AAC CAA TTC AGT TAT GAT AAA 1345 Leu Ser Asp Gly Ser Val He Pro Gly Asn Gin Phe Ser Tyr Asp Lys 435 440 445 TGC AGA CGG AGA TTT GAC CTG GGA GAT GCA GAA TAT TTA AGA TAC CGT 1393 Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala Glu Tyr Leu Arg Tyr Arg 450 455 460

Claims: CLAIMS 1. An am o acid sequence of starch branching enzyme II (SBE II) comprising the ammo acid sequence as shown m SEQ ID No. 1. 2. Fragments of the ammo acid sequence of starch branching enzyme II (SBEII) . 3. A fragment according to claim 2, having the ammo acid sequence as shown in SEQ ID No. 2. . An isolated DNA sequence encoding s1 arch branching enzyme II (SBE II) of potato comprising the nucleotide sequence as shown m SEQ ID No. 1 variants 1 hereof resulting from the degeneracy of the genetic code. 5. Fragments of the isolated DNA sequence encoding starch branching enzyme II (SBEII) of potato. 6. A fragment according to claim 5, comprising the nucleotide sequence as shown in SEQ ID No. 1 . 1 . A vector comprising the whole or a functionally active part of the isolated DNA sequence claimed in any one of claims 4-6 and regulatory elements active in potato. 8. A vector according to claim 7, wherem the DNA sequence is m the antisense (reversed) orientation m relation to a promoter immediately upstream from the DNA sequence. 9. A process for the production of transgenic potatoes with either an increased or a decreased degree of branching of amylopectin starch, c h a r a c t e r i z e d m that it comprises the following steps: a) transfer and incorporation of a vector c ccordιng to claim 7 into the genome of a potato cell, nd b) regeneration of intact, whole plants from the transformed cells. 10. A process for the production of ti ansgenic potatoes with a reduced degree of branching of amylopectin starch, c h a r a c t e r i z e d m that it comprises the following steps: a) transfer and incorporation of a vector according to claim 8 mto the genome of a potato cell, and b) regeneration of intact, whole plants from the transformed cells. 11. A process according to claim 10, wherein the vector also comprises an antisense construct of starch branching enzyme I (SBE I) . 12. A process according to claims 10 or 11, wherein the vector also comprises an antisense construct of potato granule bound starch synthase II. 13. A process according to one or more of claims 10-12, wherein the vector also comprises an antisense construct of potato soluble starch synthases II and III. 14. A process according to one or more of claims 10-13, wherein the vector also comprises an antisense construct of potato starch disproportionatmg enzyme (D- enzy e) . 15. A process according to one or more of claims 10-14, wherein the vector also comprises an antisense construct of potato starch debranchmg enzyme. 16. A transgenic potato obtainable by the process according to any one of claims 9-15. 17. Use of transgenic potatoes accordmg to claim 16 for the production of starch.

Inventors: EK BO, KHOSNOODI JAMSHID, LARSSON CLAS-TOMAS, LARSSON HAAKAN, RASK LARS, EK, BO, KHOSNOODI, JAMSHID, LARSSON, CLAS-TOMAS, LARSSON, HAAKAN, RASK, LARS
Applicants: AMYLOGENE HB, EK BO, KHOSNOODI JAMSHID, LARSSON CLAS TOMAS, LARSSON HAAKAN, RASK LARS, EK, BO, KHOSNOODI, JAMSHID, LARSSON, CLAS-TOMAS, LARSSON, HAAKAN, RASK, LARS
Application year: 1996
Main classification: C12N9
IPC codes: A01H5, C12N5, C12N9, C12N15