AU725657B2 - DNA sequences which lead to the formation of polyfructans (levans), plasmids containing these sequences as well as process for preparing transgenic plants - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION r r r r Name of Applicant: Address of Applicant: Actual Inventor(s): Address for Service: Hoechst Schering AgrEvo GmbH Miraustrasse 54, D-13476 Berlin, Germany ROBER, Manuela GEIER, Gebhardt GEIDER, Klaus WILLMITZER, Lothar DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete Specification for the invention entitled: DNA sequences which lead to the formation of polyfructans (levans), plasmids containing these sequences as well as a process for preparing transgenic plants The following statement is a full description of this invention, including the best method of performing it known to us: -1- 1A Title: DNA sequences which lead to the formation of polyfructans (levans), plasmids containing these sequences as well as a process for preparing transqenic plants Field of the invention The present invention relates to DNA sequences which lead to the formation of polyfructans (levans), as well as a process for preparing transgenic plants using plasmids on which these DNA sequences are located.

High molecular weight, water soluble, linear polymers, for example those based on polyacrylates or polymethacrylates, are products of mineral oils and have many important uses.

In particular their properties in increasing viscosity of aqueous systems, in suspending or sedimentation acceleration and complexing are especially valuable from the technical viewpoint. These products are also used in 20 exceptionally large amounts in super absorbers for water binding and in water dilutable lacquers. In spite of the outstanding positive properties, because such products are difficult to dispose of, their use is increasingly coming under criticism because they are not biodegradable.

Alternatives based on recyclable raw materials, especially starches and cellulose, because of the macromolecular structure of these polysaccharides, have been shown to have limited value. As a replacement for non-biodegradable 30 chemically derived polymers, a number of derivatised high polymeric polysaccharides have been considered. Until now, such polysaccharides could only be obtained biotechnologically via suitable fermentation and transglycosidation processes. The products obtained in this way, such as dextrans and polyfructans (levans) are 2 not competitive as raw materials for mass production.

Polyfructans are found in a number of monocotyledonous and dicotyledonous higher plants, in green algae as well as in a number of gram positive and gram negative bacteria (Meier and Reid, (1982) Encyclopedia of Plant Physiology, New Series, 13A. 418 471). The role of fructans for the plant development and plant growth is not fully understood. Functions of the fructans that have been proposed are as a protection against freezing at low temperatures, as alternative carbohydrate stores by limiting starch biosynthesis, as well as applied intermediary stores for photoassimilates, situated in the stems of grasses, shortly before their transfer into the seeds.

All fructans contain as starter molecule for the polymerisation reaction, a molecule of sucrose (glucosefructose) to which fructose polymers are added.

2 Depending on the coupling of the fructose molecule, fructans of plant origin can be classified into four classes (Meier and Reid (1982), Encyclopedia of Plant Physiology, New Series, 13A, 418 471): a) coupled f-D-fructans (inulin type) b) coupled O-D-fructans (phlein or levan type) 30 c) highly branched fructans with a mixture of 2-1 and 2- 6 couplings.

d) coupled P-D-fructans, which in contrast to the types under a c, are added completely from fructose residues of polymerisation both from glucose and also from fructose residues from polyfructose residues (neokestose type).

Fructans of bacterial origin correspond either to the levan or to the inulin type (Carlsson (1970) Caries Research 4, 97 113) and Dedonder (1966) Methods Enzymology 1, 500 505).

Experiments on the biosynthesis of fructans in plants and bacteria lead one to conclude that this proceeds by various routes. Bacterial and plant fructans are further distinguished, not particularly in their primary structure but mainly in their molecular weight. Thus, fructans isolated from plants have been shown to have molecular weights of between 5000 and 50,000 d (Pollock and Chatterton (1988) in: The Biochemistry of Plants 14, 109 140), whilst for fructans isolated from bacteria, molecular weights of up to 2,000,000 d have been described (Clarke et al (1991) in: Carbohydrates as Organic Raw Materials, VCH Weinheim, 169 182).

Various microorganisms from the group of Bacillus spp as well as Streptococcus spp produce polyfructoses in which both fructans of the levan type and fructans of the inulin 25 type have been described (Carlsson (1970) Caries Research A, 97 113 and Dedonder (1966) Methods Enzymology 8, 500 505).

Experiments on biosynthesis pathways have made it clear 30 that, in comparison to biosynthesis pathways in higher plants, there is a more simple pattern and a sharing of only one enzyme. This enzyme with the trivial name levan sucrase is a transfructosylase (sucrose:B-D-fructosyl transferase, which catalyses the following reaction: sucrose acceptor glucose fructosyl acceptor Representative acceptors are water, alcohol, sugar or polyfructoses. The hypothesis that only one enzyme catalyses this reaction, depends on the one hand on the examination of the protein chemically purified enzyme, and on the other to the fact that the gene for levan sucrase has been isolated from various Bacillus spp. as well as from a Streptococcus spp. and after transfer into E. coli leads to the formation of levan in E. coli (Gay et al (1983) J. Bacteriology 153, 1424 1431 and Sato et al.

(1986) Infection and Immunity 52, 166 170).

Until now, genes for levan sucrase from Bacillus amyloliquefaciens (Tang et al. (1990) Gene 96, 89 93) and Bacillus subtilis (Steinmetz et al. (1985) Mol. Gen.

Genetics 200, 220 228), have been described, which demonstrate relatively high homology with each other and both of which catalyse the synthesis of fructans of the 20 levan type. Further a fructosyl transferase from Streptococcus mutans (Shiroza et al. (1988) J.

Bacteriology 170, 810 816) has been described. This shows little homology to either levan sucrases from Bacillus spp.. The fructan formed in Streptococcus mutans 25 is of the inulin type.

In WO 89/12386, there is described the possibility of producing carbohydrate polymers such as dextran or levan in transgenic plants, especially in the fruit of transgenic plants. To prepare these plants, the use of levan sucrases from Aerobacter levanicum, Streptococcus salivarius and Bacillus subtilis and the use of dextran sucrases from Leuconostoc mesenteroides have been described.

Further the construction of chimeric genes is described which may be suitable for the expression of the levan sucrase from Bacillus subtilis as well as the dextran sucrase from Leuconostoc mesenteroides in transgenic plants. Also described is the preparation of transgenic plants containing these constructs. Further, the preparation of transgenic plants that contain these constructs are described. Whether polyfructans can actually be produced by the described process is not known.

There is also described a series of processes for modifying the carbohydrate concentration and/or concentrating carbohydrate in transgenic plants by means of biotechnological methods. Thus, in view of the fact that increasing starch concentration and modification of the starch in physical and chemical respects is already known, then a modification of the carbohydrate content of potato plants by raising or lowering the ADP-glucosepyrophosphorylase activity can be achieved (EP 455 316).

From EP 442 592 it is further known that a modification of the distribution of photoassimilates by means of cytosolic and apoplastic invertase is. possible and that the yield as 25 well as the drought and frost resistance of potato plants through expression of a heterologous pyrophosphatase gene in potato plants can be modified.

In order to adapt the physico-chemical parameters of raw 30 materials which are increasingly being used, such as polysaccharides, to the requirements of the chemical industry, as well as to minimise the costs of obtaining these products, processes for the preparation of transgenic plants have to be developed which lead in comparison with known processes to better, higher yielding plants.

6 It has now been surprisingly found that the DNA sequence of the levan sucrase from a gram-negative bacterium of the species Erwinia amylovora with the nucleotide sequence (Seq -ID NO 1): GGATCCCCCG GGC-TGCAGCG ATCATGGTTA TTTATAAGGG ATTGTTA7GT CCTGAAAACC ACACAACAGA ACCAGAGTGA

TTTCLAAAAA

TTA ATAT1.1ACA GACCTTCAGC PLAGAAGGTAT

'TCGAAATAA-C

ATTT ATG TCA GAT Met Ser Asp

TAAAAAGCTA

CTGTGAGGAT

100 150 163 TAT A-AT TAT AA. CCA

S

S

S S Tyr

GTT

Val

GCA

Al a

CCA

pro

TGT

Cys

CAA

GIn

GA

Glu

ACC-

Thr

GTC

Val

AAC

Asn

GGT

Gly

CAA

Gin

TCT

Ser Asn

CAT

His

TTC

Phe

TTG

Leu

ATT

Ile 65

TTC

Phie

GAC

Asp 95

GGT

Glv

G'CA

Ala 125

GAT

ASO

140 G CA Al a 155

AGT

Ser 170

GCT

Ala 185 Tyr

GAG

Glu

CCG-

Pro

CGA

Arg

ATT

Ile

CAG

Gin

AGA

Arg AXkA Lys

CCG

Pro

CGG

Arg Thr

GTA

Val

GXC

Asp Lys

GAT

Asp)

GTA

Val

GAC

Asp

TTT

Phe

GAT

AspD

CAT

His

GAC

Aspo

ACG

Th Z

GGC

Giy

ATT

Ile

AGC_

Ser

GGG'

Gly Pro

GAC

Asp

ATG

Me t

TTC

Phe

ACG

Thr

GAA

Glu

GGT

Gly

TGG

Tzr,

ACG

Thr

GAT

Asti

GCC

Ala

CTG

Leu

ACT

Thr

ACG

Thr

CCA

AGT

Ser

GAC

Asp

CTA

Lau

AAT

Asn

CGT

Ar g

ATT

CGT

xrg Lys

C-AA

G_'u

ATT

Ile CTG TGG ACT Lau Trp Thr 10 ACC ACA ACT- Thr Thr Thr 25 GAA GAA GTC Glu Glu Val.

40 GGA GAG ATT Gl1v Glu lie 55 ACAN GCA GAT Thr Ala Asp 70 G GC AAT TAT Gly Asn Tyr 859 GCGCGT

ATT

Ala Arg Ile 100 TTT GGC GG T Phe Gly Glv 115 GAG TGG G%C Glu Trp. Ala 10 GAC CTG TAT Asp Leu Tyr.

145- GT G C GGT Val Arg Gly 160 GGT TTT CAG Giv Phe Gin 175) TAC CAG ACG Tyr Gin Thr 190

CGT

Arg

CAA

Gin

TTT

Phe

ATC

le

CGC

Arg

GAT

Asp

TGT

Cys CG G Arg

GGA

Gly

TAT

Tyr

AAA

Lys

CAG

Gin

GAA

GCC

Al a

CCG

Pro

ATT

Ile

TCT

Ser

AAC

Ash

ATT

Ile

TAT

Tyr

GTA

Val

ACC

Thr

ACC

Thr

ATC

le

GTT

Val

GAG

GAT GCA Aso Ala GTT ATT Val le T GG GAT Trp -Aso GTA AAT Val Asn ACT GAT Thr Asp ACT CGT Thr Arg TGG TAC Trp Tyr 105 ATG G C C Met_ Ala 120 CCG ATC Pro !!a 135 TGT GTC Cys Val 150 GTC ACT Val Thr 16'5 ACA T CA.

Thr Se-_ 180 T TG AAA L eu Ly s 208 GAC ATT 253 AsZ T Ie ACC ATG Thr %Iet% 298 GGT TGG33 Giy T=p AAT CCG 388 Asn Pro GAC TGG-I Asp TrP 433 TCA CGC 478 Se a rg GAA GGT 523 Glu Giy CTT TTA Leu Leu 568 ACT C CG 613 T hr Pro TCC G AT 658 CTT TTC Lau Phe 703

S

CG AsA a G PheC74 Glu Giu 195 Asn Ala ?he TGG AAC Trp Asn 200 AA TTA Lys Leu 215 TCG CAC Ser His 230 TAT GAA Tyr Glu 245 CTG GCT Lau Ala 260 CCT CCG Pr--o Pro 275 CCT CAT Pro His 290 A GC CAT Ser His 305 GTG TAT Val Tyr 320 ATG AAT Me t Asn 335 TTC GAG Phe Gin 350 TCC TTT Ser Phe 365 GGC GG%3T Giv Giv 380 CGC TCA Arg Ser 395 ATG AAA Met Lvs 410 TTC CGT GAC Phe A=-g Aso

TAT

Tyr ATG CTG Met Leu GAA ATT ACC C-lu lie Thr GAT GTG GGT Asp Val Giy GTG GCC AAA Val Ala Lvs CTG ATC ACC Leu lie Thr TTT GTC TTC Phe Val Phe AAG TAG ACT Lys Tyr Thr CCA AGC CCA Pro Ser Pro 205 TTT GAA GGA Phe Giu Gly 220 CAG GGT GAG Gin Ala Glu 235 GGC GCA AA.A Gly Ala Lys 250 GAG CTG TGA AsD Lau Ser 265 GCT GTT G G C Ala Val. Giv 280 GAG GAT GGT Gin Asp Gly 295 TTT GCC GAT Phe Ala Asp 310 AGC GAT AA Ser Asp Lys 325 CTG, GTG CTG Leu Val Leu 340 GAG TAT GTT His Tyr Val 355 GTT CGG TGG Val Pzro.TrD 370 TTC ATT QAC A GGC Phe lle Asp Arg 210 AAC GTG GCG GGG Asn Val Ala Glv 225 ATG GGT AAT GTG Met Gly Asn Val 1240 TAT GAG GCA GGC Tyr Gln Ala Glv 255 GGC AGT GAG TGG Gly Ser Giu T-rm 270 GTA AAC GAT GAG Val Asn Asp Gin 285 AAA TAC TAT CTG Lys Tyr.Tyr- Leu 300 AAC CTG ACC GGC Asn Lau Thr Gly 315 CTT ACC GGC GCT Leu Thr Gly Pro 330 GGG AAC CCG TGT Giy Asn Pro Ser 345 ATG CCT AAT GGG Me t Pro Asn Glv 360 AAA GGT AJAG GAG Lys Gly Lys Asp 375 A-AT GAT GGC Asn Asp G'Ly 793 CCG CGG GGT Pro Pro Glv TGT GTT GGT Cvs Val. Giv 883 928 CC G CGSC GGT 3 Pro Arg Gly83 CAA ATC CTG97 Gin IeLau ACT GAA CG%-C 1 0 1 8 Thr Glu Arg TTC ACC ATT Phe Th= -ie 1063 CGCT GAT GGA 1 1 0 8 Pro Asp Gly TAG ACG CCG, Tr Thr Pro 1153 TCA CAA C C.T 1198 Ser Gin Pro GTG GTG ACT 1243 Leu Val Thr TAT CGC ATT T yr ArC TI- 1288 I~

C

*0

C

(C C GGC TTT Gly Phe AGC TCC Ser Ser ACA TAT Thr Tyr ATT GAG le Asp

GTA

Val

GGG-

Gly

TCA

Ser

AGT

Ser ACT GAA GCT CCG Thr Glu Ala Pro TTT ATT GTT GAkT Phe le Val. Asp GAC ATT ACT TTA Asp lie Thr Lau ACC GTA AAA ATT CTG TTG Thr? Val Lys Tle Leu Leu 385 390

AAA

Lys

GGC

Gly GAT 13 Asp 33 AGC TTC GAT TAT GGA TAT'-ATT Ser Phe Asp T-yr Gly Tyr le 400 405 CCG GC-a. 1378 Pro Ala AAA TAAiGTCTGTT GTCGATATCA AGCTTATCGA'1429 Lys 415 TACCGTCGA' 1438 makes possible the preparation of large amounts of polyfructans (levans) in transgenic Plants, which decisively meet the needs of the chemical industry in respect of recyclable raw materials.

By integration of a DNA sequence in a plant genome, on which the above given DNA sequence is located, the polyfructan (levan), expression in plants, especially in, leaves and tubers is made possible. -The-levan sucrase of the invention shows, at the DNA level, no significant homology to the known levan sucrases.

The invention further provides a process for the preparation of transgenic plants with polyfructan (levan) expression in leaves and tubers that comprises the following steps: preparation of a DNA sequence with the following partial sequences: i) a promoter which is active in plants and ensures formation of an RNA in the intended target tissues or target cells, ii) a DNA sequence of a levan sucrase, and iii) a 3'-non-translated sequence, which in plant 20 cells leads to the termination of the transcription as well as the addition of poly A residues to the 3'-end of the RNA, transfer and integration of the DNA sequence in the plant genome of a recombinant double stranded DNA 25 molecule from plant cells using a plasmid, and regeneration of intact whole plants from the transformed plant cells..

The levan sucrose obtained in process step ii) 30 preferably shows the nucleotide sequence noted under sequence IC No 1.

The levan sucrase catalyses the following reaction: Sucrose-(fructose)n sucrose sucrose-(fructose),n+ glucose.

Using this process in principle, all plants can be modified in respect to a polyfructan (levan) expression, preferably crops such as maize, rice, wheat, barley, sugar beet, sugar cane, tobacco and potatoes.

In process step in principle all plasmids can be used which have the DNA sequence given under sequence ID No 1.

Preferably used are plasmid p35s-CW-LEV (DSM) 7186), plasmid P35s-CY-LEV (DSM 7187) or plasmid P33-CW-LEV (DSM 7188).

Since sucrose represents the substrate for the levan sucrase, the production of polyfructans is especially advantageous in those organs which store large amounts of sucrose. Such organs are for example the roots of sugar beet or the stems of sugar cane. It is especially useful 20 in genetically modified potatoes, which store sucrose in their tubers, through blocking of starch biosynthesis Biosynthesis of sucrose takes place in the cytosol, whilst in contrast, storage is in the vacuole. During transport 25 into the storage tissues of sugar beet or potato or into the endosperm of seeds, the sucrose must cross the intercellular space. In production of polyfructans, all three cell compartments are suitable, i.e. cytosol, vacuole and intercellular space.

The coding sequence of the levan sucrose of the nucleotide sequence ID No 1 can be provided with a promoter that ensures the transcription in specified orders which is coupled in sense orientation (3'-end of the promoter to the 5'-end of the coding sequence) on the coding sequence which codes the enzyme to be formed. The termination signal which determines the termination of the mRNA synthesis is adhered to the 3'-end of the coding sequence.

In order to direct the enzyme which is expressed in specified sub-cellular compartments such as chloroplasts, amyloplasts, mitochondria, vacuoles, cytosol or intercellular space, a so-called signal sequence or a transit peptide coding sequence can be positioned between the promoter and the coding sequence. This sequence must be in the same reading frame as the coding sequence of the enzyme.

For the introduction of the DNA sequence of the invention 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, 20 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 25 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, &00;,0 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) 0 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.

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 be tested 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 20 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 81-84). These plants can be grown normally and crossed with plants, that possess the same transformed genes or 25 different. The resulting hybrid individuals have the corresponding phenotypical properties.

Deposits The following plasmids were deposited at the Deutschen 30 Sammlung von Mikroorganismen (DSM) in Braunschweig, Germany on the 16.07.1992 (deposit number): Plasmid p35s-CW-LEV (DSM 7186) Plasmid p35s-CY-LEV (DSM 7187) Plasmid p33-CW-LEV (DSM 7188) Description of the Figures Fig. 1 shows the structure of the p35-CW-LEV plasmid.

It comprises the three fragments A, B and C.

Fragment A contains the 35s promoter of the cauliflower mosaic virus (CaMV), nucleotides 6906 7437.

Fragment B contains the sequence of the nucleotides 689 2122 of the levan sucrase from Erwinia amylovora (Seq. ID No.1).

Fragment C contains the polyadenylation signal of the gene 3 of the T-DNA of the Ti-plasmid, pTi ACH 5, nucleotides 11749 11939.

Fig. 2 shows the structure of the p35s-CY-LEV plasmid.

It comprises the three fragments A, B and C.

Fragment A contains the 35s promoter of the cauliflower mosaic virus (CaMV), nucleotides 6909 7437.

Fragment B contains the sequence of the nucleotides 864 2122 of the levan sucrase from Erwinia amylovora (Seq. ID No.1).

Fragment C contains the polyadenylation signal of the gene 3 of the T-DNA of the Ti-plasmid, pTi ACH Fig. 3 shows the structure of the p33-CW-LEV plasmid.

It comprises the three fragments A, B and C.

Fragment A contains the DraI-DraI-fragment (position -1512 to position +14) of the promoter region of the patatin gene B33.

Fragment B contains the sequence of the nucleotides 689 2122 of the levan sucrase from Erwinia amylovora (Seq. ID No.l).

Fragment C contains the polyadenylation signal of the gene 3 of the T-DNA of the Ti-plasmid, pTi ACH 5, nucleotides 11749 11939.

P:\OPER\MRO\49468-93.CLM 9/1/98 -13- Fig 4. shows the detection of polyfructan in transformed tobacco plants (No. 2, 3 and 13).

In this: Fru fructose, Suc sucrose, Kes kestose cl control 1, c2 control 2, M marker Fig 5. shows 2,6-D-Fructofurane(levan) synthesized in transgenic plants by 13C NMR spectroscopy.

In this: numbering on the x-axis and at the top of the figure indicate ppm in sample.

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 The vector pUC 18 (Yanisch-Perron et al. (1985) Gene 33: 103-119) was used for cloning.

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 25 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 vectors. TB1 is a recombinant-negative, tetracycline-resistant derivative of strain JM101 (Yanisch-Perron et al., Gene (1985), 33, 103-119). The 30 genotype of the TB1 strain is (Bart Barrel, personal communication): F'(traD36, proAB, lacI, lacZAM15), A(lac, pro), SupE, thiS, recA, Srl::TnlO(TcR).

o' 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 Aarobacterium tumefaciens 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 A) Tobacco: 10 ml of an overnight culture of Agrobacterium tumefaciens, grown under selection, were centrifuged off, the supernatant was discarded, and the bacteria were resuspended in the same volume of antibiotic-free medium.

In a sterile petri dish, leaf discs of sterile plants (approximately 1 cm 2 the central vein of which had been removed, were immersed in this bacterial suspension. The leaf discs were then placed in a closely packed arrangement in petri dishes containing MS medium (Murashige et al. (1962) Physiologia Plantarum 15, 473- 497) with 2% sucrose and 0.8% bacto agar. After two days incubation in the dark at 250C, they were transferred onto MS medium containing 100 mg/1 kanamycin, 500 mg/l claforan, 1 mg/l benzylaminopurine (BAP), 0.2 mg/l of naphthylacetic acid (NAA) and 0.8 bacto agar. Growing shoots were transferred onto hormone-free MS medium with 250 mg/l of claforan.

B) Potato; 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 ~1 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/i of zeatin ribose, 0.02 mg/1 of naphthylacetic acid, 0.02 mg/l of gibberellic acid, 500 mg/1 of claforan, 50 mg/1 of kanamycin and 0.8% bacto agar. After incubation for one week at 25 0 C and 3000 lux, the claforan concentration in the medium was reduced by half. Further cultivation was carried out using the method described by Rocha-Sosa et al. (1989) EMBO Journal 8, 29).

Analysis of genomic DNA from transqenic plants The isolation of genomic plant DNA was carried out according to Rogers et al. (1985) Plant Mol Biol 69-76).

For the DNA analysis, after suitable restriction cleavage, 10 to 20 pg of DNA were analysed, by means of Southern blotting, for the integration of the DNA sequences to be investigated.

6. Analysis of the total RNA from transqenic plants 20 The isolation of plant total RNA was carried out according to Logemann et al. (1987), Analytical Biochem.

.163. 16-20.

For the analysis, 50 pg portions of total RNA were investigated, by means of Northern blotting, for the presence of the transcripts sought.

7. Extraction and determination of polyfructose in plants The extraction and determination were carried out according to the method of Portis H. G. (1990), Meth.

Plant Biochem. 2, 353-369.

Example 1 Preparation of plasmid p35s-CW-LEV and insertion of the plasmid into the qenome of tobacco and potato The plasmid p35s-CW-LEV comprises the three fragments A, B and C, which were cloned in the cutting sites for restriction enzymes of the polylinker from pUC 18 (see Fig. 1).

Fragment A contains the 35S promoter of cauliflower mosaic virus (CaMV). It contains a fragment that includes the nucleotides 6909 to 7437 of CaMV (Franck et al. (1980) Cell 21, 285-294) and was isolated as Eco RI-Kpn I fragment from plasmid pDH 51 (Pietrzak et al., Nucleic Acids Research 14, 5857-5868) and cloned between the Eco RI-Kpn .I cutting sites of the polylinker of plasmid pUC 18.

Fragment B contains the sequence of the nucleotides 689 20 2122 of the gene of the levan sucrase from Erwinia amylovora (Seq. ID No.l) and was cloned between the BamHI/SalI cutting positions of the polylinker of pUC 18.

Fragment C contains the polyadenylation signal of the gene 25 3 of the T-DNA of the Ti-plasmid, pTi ACH 5 (Gielen et al (1984); EMBO J. 3, 835 846) nucleotides 11749 11939 which was insolated as Pvu II-Hind III fragment from the plasmid pAGV 40 (Herrera-Estrella et al (1983) Nature 303, S. 209 213) and, after addition of Sph I linkers to the Pvu II cutting positions, was cloned between the SphI-Hind III cutting positions of the polylinker of pUC 18.von pUC 18.

The plasmid p35s-CW-LEV has a size of 2151 bp.

The part of the plasmid p35s-CW-LEV comprising the fragments A, B and C was introduced in binary vectors and 1.

17 using the Agrobakteria system was introduced into tobacco and potato plants. Intact plants were regenerated from transformed cells. The analysis of the leaves from a series of Tobacco plants transformed with this gene, clearly showed the presence of polyfructan (levan) which is traced back to the expression of the gene (see Fig. 4).

Example 2 Preparation of plasmid p35s-CY-LEV and insertion of the plasmid into the genome of tobacco and potato This Example was carried out in an analogous manner to that described under Example 1, but with the modification, that the Fragment B (coding for the levan sucrase) is shortened on the nucleotide at the 5'-end. This results in the expression of the protein in the cytosol of transgenic plants.

20 The plasmid p35s-CY-LEV comprises the three fragments A, B and C, which were cloned in the cutting sites for restriction enzymes of the polylinker from pUC 18 (see Fig. 2).

Fragment A contains the 35S promoter of cauliflower mosaic virus (CaMV). It contains a fragment that includes the nucleotides 6909 to 7437 of CaMV (Franck et al. (1980) Cell 21, 285-294) and was isolated as Eco RI-Kpn I fragment from plasmid pDH 51 (Pietrzak et al., Nucleic Acids Research 14, 5857-5868) and cloned between the Eco RI-Kpn I cutting sites of the polylinker of plasmid pUC 18.

Fragment B contains the. sequence of the nucleotides 864- 2122 of the gene of the levan sucrase from Erwinia amylovora (Seq. ID No.l) and was cloned between the SmaI/SalI cutting positions of the polylinker of pUC 18.

Fragment C contains the polyadenylation signal of the gene 3 of the T-DNA of the Ti-plasmid, pTi ACH 5 (Gielen et al (1984); EMBO J. 3, 835 846) nucleotides 11749 11939 which was insolated as Pvu II-Hind III fragment from the plasmid pAGV 40 (Herrera-Estrella et al (1983) Nature 303, 209 213) and, after addition of Sph I linkers to the Pvu II cutting positions, was cloned between the SphI-Hind III cutting positions of the polylinker of pUC 18.von pUC 18.

The plasmid p35s-CY-LEV has a size of 1976 bp.

The part of the plasmid p35s-CY-LEV comprising thefragments A, B and C was introduced in binary vectors and using the Agrobakteria system was introducedinto tobacco and potato plants. Intact plants were regenerated from transformed cells.

20 Example 3 Preparation of plasmid p35s-CY-LEV and insertion of the plasmid into the qenome of tobacco and potato This Example was carried out in an analogous manner to 25 that described under Example 1, but with the 35s promoter being replaced with the promoter of the class I patatin Gene B33 (Rocha-Sosa et al, (1989) EMBO J 8, 23 29) The plasmid p33-CW-LEV comprises the three fragments A, B and C, which were cloned in the cutting sites for restriction enzymes of the polylinker from pUC 18 (see Fig. 3).

Fragment A contains the Dral-Dral fragment (position -1512 to position +14) of the promoter region of the patatin gene B33 (Rocha-Sosa et al (1989) EMBO J. 8, 23 29), I W 1, 19 which was cloned in the Sma I position of the polylinker of pUC 118.

Fragment B contains the sequence of the nucleotides 689-2122 of the gene of the levan sucrase from Erwinia amylovora (Seq. ID No.l) and was cloned between the BamHI/SalI cutting positions of the polylinker of pUC 18.

Fragment C contains the polyadenylation signal of the gene 3 of the T-DNA of the Ti-plasmid, pTi ACH 5 (Gielen et al (1984); EMBO J. 3, 835 846) nucleotides 11749 11939 which was insolated as Pvu II-Hind III fragment from the plasmid pAGV 40 (Herrera-Estrella et al (1983) Nature 303, 209 213) and, after addition of Sph I linkers to the Pvu II cutting positions, was cloned between the SphI-Hind III cutting positions of the polylinker of pUC 18.von pUC 18.

The plasmid p33-CW-LEV has a size of 3149 bp.

The part of the plasmid p33-CW-LEV comprising the 20 fragments A, B and C was introduced in binary vectors and using the Agrobakteria system was introduced into tobacco and potato plants. Intact plants were regenerated from transformed cells. The analysis of the leaves from a series of Tobacco plants transformed with this gene, clearly showed the presence of polyfructan (levan) which is traced back to the expression of the gene 33-CW-LEV.

a oo Example 4 Analysis of p2,6-D-Fructofurane (levan) synthesised in transgenic plants by 13C-NMR spectroscopy The analysis of transgenic plants transformed with the construct p35s-CW-LEV is shown as an example. This analysis can equally be applied to transgenic plants transformed with the constructs p35S-CW-LEV or To obtain sufficient amounts of levan synthesised by transgenic plants to perform NMR spectroscopy, about 10g of leave tissue were grinded in 10ml of water. The homogenate is than centrifuged at 4000 Rpm in a Beckman Minifuge and the supematant is applied to a column (LKB-Pharmacia) to remove lower molecular weight compounds. The column had been equilibrated with water before 2.5 ml of the supernatant are applied and higher molecular weight compounds are then eluted with 3.5 ml of water. The elute was further purified by adding ion exchange beads (AG 501 X8, Biorad) and shaking for 30 minutes.

After centrifugation at 4000 Rpm (Minifuge, Beckman) to remove the beads, the supernatant is applied to a Sepharose 4B column (diameter 16 cm, separating volume 24 ml) to remove short sugar chains. The elute is vacuum dried in a vacuum centrifuge (univapo 150 H, Uniquip, Martinsried (FRG) and than analysed by 13C-NMR under the following conditions: PULPROG zgdc30 F2 Processing parameters SOLVENT D20 SI 32768 AQ 1.3762726 sec SF 100.5485322 MHz FIDRES 0363305 Hz WDW EM DW 21.0 usec SSB 0 RG 32768 LB 0.50 Hz S NUCLEUS 13C GB 0 Dll 0.0300000 sec PC 1.40 P31 100.0 usec S2 20 dB 10 NMR plot parameters HLI 1 dB CX 33.00 cm D1 1.0000000 sec F1P 123.000 ppm P1 6.5 usec F 12367.47 Hz DE 263 usec F2P -6.000 ppm SF01 100.5597430 MHz F2 -603.29 Hz SWH 23809.58 Hz PPMCM 3.90909 ppm/cm TD 65536 HZCM 393.05334 Hz/cm NS 8000 DS 2 The result of the analysis is shown in Fig. 5. The pattern of NMR peaks obtained is the same as it is obtained for levan as published by Gross et al., 1992, Physiol Mol Plant Pathol 371.

This proves that the transformed plants synthesise levan after transformation by one of the constructs described in examples 1 to 3.

21 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF TIM DEPOSIT OF MICROORGA~nS FOR THE PURPOSES OF PATEN1T PROCEDURE mIERNATioNAL FORm Institut ff= Genbiologische Forschuig Berlin GmbH Thnestr. 63 W-1000 Berlin 33 VIADITY STATUIT issued pursuant to Rule 10.2 by tdo ITERNATIONAL DEPOSITARY AUTHORITY idtfied at th. bottom of this page a

C

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1. DEPOSITOR IL WENTWiOATiON OF THE MICROORGANISM Nam Institut ffir Genbiologische Amsosm number givan by the Forschung Berlin GmbH INTERNATIONAL DEPOSITARY AUTHORITY: Addres: Thnestr. 63 DM78 W-1000 Berlin 33 DN78 Dabe of the deposit or of the transfer 2 1992-07-16 131 VIABIRiT STATEMENT The viability of the microorganism identified unde U aho, watested on 1992-07-17 *2 On that data, the said microorganism was (X )3 viable no loger viable IV. CONDITIONS UNDER WHICH THE VIADITY TEST HAS BEEN PERFORME 4 IV. INTERNATIONAL DEPOSITARY AUTHORITY Name: DSM DEUTSCHE SAlOLLUNO VON Signature(s) of pemsn(s) having the powea JVUIOOORGANLSMN UND ZELLKULTUREN GmbH to repreent the International Depositary Authority or of authorised official(s): Address: Mamheroder Weg I13 D-W30 BraunschweigC- L e 4 Dat-e.' 992-07-21 1Indicate the date of originial deposit or, where a now deposit or a tnaser has been made, the most recent relevant date (date of Wh new deposit or date of the transfe).

2 In thecases referred toin Rule 1.2(s) (it) and refer to the mot recent vablity teet.

3Mark with a cross the applicable box.

4Fill In if the informnation has been requested end If the reslts of the test were negative.

'a -C Form DSM-BP/9 (sole page) 0787 I i 0 I 22 BUDAPESTER VERTRAG 10BER DMR DMENAIONA1T jAlERIC2(NUNG DER NDIERLEGWIG VON MKOORGANLSMN FOR DIE ZWECKE VON pALTENTYZEPANREN DITERNATioNALEs FORmELATT Institut far GenbiolOgiScile Porschung Berlin GmbH Ihnestr. 63 W-1000 Berlin 33 EMPTANGS13ESTALTIGUNG BRE-T I RMERUUNG, anagestelit gamES Rage 7.1 va der unten angegabanim I MNATIONALEN E!NTERLEGUNCSSTELLE 'a e..a* a a a L ENNZEJCENUNG DES MnOORGA1SMUS Yarn H TERLEGER zugatailtas Besupsaichan: Van der UNTERNATIONALEM ETERLEGUNC SSTELL sugetsilts EINGANGSNUhGdERp33 -CW-Lev DSM 718 M. WISSENSCHArLICHE BESCEREIDUNG UND/ODF.R VOB.GESCELAGENE TAXONOMISCRE BEZEICEIANG )At dam unter L beichnoten Mikzuaorganismus vuida ins wisvenscha fticba Bschweibung )in vo urgeschlagene taxonommsche Bomichnung (Zutrffooan ankreusan).

13. EINGANG UND ANNABME Dine intemnationale Kintaulagungsstelle imzmt den untar I bessichnatan Mikuoarganismus an, der bei ibm am 1992-07-16 (Datum der Ersthinterlegung) 1 eingegangmn ist.

IV. EDIGANG DES AIITRAGS AUF UMWANDLUNG Der unter I beseichnets Mikmoorganismus ist bal diaee Intarustionalen Rinterlegungutelie am eingegangen (Datum der Ersthintarlegung) und tin Antrag auf Umwandlung dinser Enthintedegung in ein. Hintaulagung geniE Budapaer Vertrag Ist am sngangen (Datum des Elagazp des Antrap auf Urnwandlung).

V. DITERNATIONALE maNTERLEGUNGSSTELLE Nlama: DSM-DEUTSCHE SAW04UNG VON Unterochrift(an) der sur Vests tung der intarnationalen QIM ORGANISUEN UND ZELLKULTUREN GmbH Htintauagunpotal. befugton Parson(en) oder des (der) van ihr ermichtigten Befienstaten Adwesac: Macherodar Wag 1 B 7 Braunschweig Datum: 1992-07-21 SFabe lege! 6.4 Buchatabe d sutriff, ist dies der Zeitpunkt. zu dew der Status amner intemnationsan Hintaulegungnstelle arworben warden ist.

Formblatt DSM-BP/4 (einhige Seite) 0291 P:\Opr\Ej\A\MEND\1985269.hoammedcaisnm-nds /0 -23- Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

00 S 00 0 0 0000 **0 0* S. 0 0 0 0 0

Claims (7)

1. 2. The method according to Claim 1 wherein modified polyfructan formation is exclusively in non-apoplast.
2. 3. The method according to Claims 1 or 2, wherein the non-apoplast comprises chloroplasts and/or amyloplasts and/or mitochondria and/or cytosol and/or vacuoles. P:\OM\Ejh\AMENDED \I 8529.t..h.., ddl~i d-2 /0/0
3. 4. The method according to any one of Claims 1 to 3 wherein the DNA sequence encoding polyfructan (levan) sucrase is derived from Bacillus amyloliquefaciens or Streptococcus mutans. A plant obtainable by the method according to any one of Claims 1 to 4.
4. 6. The plant according to Claim 5, which is a crop plant. I* a
5. 7. The plant according to Claim 5 or 6, which is a maize, rice, wheat, barley, *0000 sugar beet, sugar cane, tobacco or potato plant.
6. 8. An isolated DNA sequence coding for a polyfructant sucrase from an organism other an Erwinia amylovora (Seq-ID No. 1) when used to produce a plant with modified polyfructan formation in non-apoplast, said plant not exhibiting a modified polyfructan formation in the apoplast.
7. 9. A method according to Claims 1-4 or a plant according to Claims 5-7 or an isolated DNA sequence according to Claim 8 substantially as hereinbefore described with reference to the Examples and/or Figures. 00 0 DATED this twenty-first day of August 2000. Hoechst Schering AgrEvo GmbH by DAVIES COLLISION CAVE Patent Attorneys for the Applicant