AU4892100A - Enhanced accumulation of trehalose in plants - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Mogen International N.V.

Actual Inventor(s): OSCAR JOHANNES MARIA GODDIJN, TEUNIS CORNELIS VERWOERD, RONNY WILHELMUS HERMANUS HENRIKA KRUTWAGEN, ELINE VOOGD Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: ENHANCED ACCUMULATION OF TREHALOSE IN PLANTS Our Ref 622257 POF Code: 128064/325678 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- ENHANCED ACCUMULATION OF TREHALOSE IN PLANTS The present application is a divisional application from Australian Patent Application Number 10085/97 (719168), the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION The invention relates to a method for the production of trehalose in plant cells, and plants. The invention is particularly related to a method for increasing the levels of trehalose accumulation in plants by inhibiting the degradation of trehalose by trehalase. The invention further comprises higher plants, preferably Angiospermae, and parts thereof, which as a result of such methods, contain relatively high levels of trehalose. The invention further relates to plant cells, plants or parts thereof according to the invention obtained after processing thereof.

STATE OF THE ART Trehalose is a general name given to D-glucosyl D-glucosides which comprise disaccharides based on two and 8,B-linked glucose molecules. Trehalose, and especially a-trehalose alpha-Dglucopyranosyl(1-1)alpha-D-glucopyranoside is a widespread naturally occurring disaccharide. However, trehalose is not generally found in plants, apart from a few exceptions, such as the plant species Selaginella lepidophylla (Lycophyta) and Myrothamnus flabellifolia. Apart from these species, trehalose is found in root nodules of the Leguminosae (Spermatophytae, Angiospermae), wherein it is synthesized by bacteroids; the trehalose so produced is capable of diffusing into the root cells.

Apart from these accidental occurrences, plant species belonging to the Spermatophyta apparently lack the ability to produce and/or accumulate trehalose.

In International patent application WO 95/01446, filed on June 1994 in the name of MOGEN International NV, a method is described for providing plants not naturally capable of producing trehalose with the capacity to do so.

In spite of the absence of trehalose as a substrate in most higher plant species, the occurrence of trehalose-degrading activity has been reported for a considerable number of higher plant species, including those known to lack trehalose. The responsible activity could be attributed to a trehalase enzyme.

2 Reports suggest that trehalose, when fed to plant shoots grown in vitro is toxic or inhibitory to the growth of plant cells (Veluthambi

K.

et al., 1981, Plant Physiol. 1369-1374). Plant cells producing low trehalase levels were found to be generally more sensitive to the adverse effects of trehalose, than plants exhibiting a higher level of trehalase activity. Trehalose-analogs, such as trehalose-amines were used to inhibit trehalase activity in shoots, making it possible to study the effects of trehalose fed to plant cells. Plant shoots which produce relatively high amounts of trehalase were adversely affected by the addition of trehalase inhibitors. Inhibition of trehalase activity in homogenates of callus and suspension culture of various Angiospermae using Validamycin is disclosed by Kendall et al., 1990, Phytochemistry 2, 2525-2582.

It is an object of the present invention to provide plants and 15 plant parts capable of producing and accumulating trehalose.

SUMMARY OF THE INVENTION The invention provides a process for producing trehalose in plant cells capable of producing trehalase by growing plant cells having the 20 genetic information required for the production of trehalose and •trehalase, or cultivating a plant or a part thereof comprising such plant cells, characterised in that said plant cells are grown, or said plant or a part thereof, is cultivated in the presence of a trehalase inhibitor.

Preferred plants or plant parts or plant cells have been genetically 25 altered so as to contain a chimeric trehalose phosphate synthase gene in a plant expressible form. According to one embodiment said trehalose phosphate synthase gene comprises an open reading frame encoding trehalose phosphate synthase from E. coli in plant expressible form.

More preferred is a gene coding for a bipartite enzyme with both trehalose phosphate synthase and trehalose phosphate phosphatase activities.

According to a further aspect of the invention, plants have been genetically altered so as to produce trehalose preferentially in certain tissues or parts, such as (micro-)tubers of potato. According to one embodiment the open reading frame encoding trehalose phosphate synthase from E. coli is downstream of the potato patatin promoter, to provide for 3 preferential expression of the gene in tubers and micro-tubers of Solanum tuberosum.

According to another aspect of the invention the plants are cultivated in vitro, for example in hydroculture.

According to another preferred embodiment said trehalase inhibitor comprises validamycin A in a form suitable for uptake by said plant cells, preferably in a concentration between 100 nM and 10 mM, preferably between 0.1 and 1 mM, in aqueous solution.

Equally suitable said trehalase inhibition can be formed by transformation of said plant with the antisense gene to a gene encoding the information for trehalase.

Also suitable as trehalase inhibitor is the 86 kD protein from the american cockroach (Periplaneta americana). This protein can be administered to a plant in a form suitable for uptake, and also it is 15 possible that the plants are transformed with DNA coding for said protein.

The invention further provides plants and plant parts which accumulate trehalose in an amount above 0.01 (fresh weight), preferably of a Solanaceae species, in particular Solanum tuberosum or 20 Nicotiana tabacum, in particular a micro-tuber of Solanum tuberosum containing trehalose.

The invention also comprises the use of a plant, or plant part, according to the invention for extracting trehalose, as well as the use thereof in a process of forced extraction of water from said plant or plant part. According to yet another embodiment of the invention a "chimaeric plant expressible gene is provided, comprising in sequence a Stranscription initiation region obtainable from a gene, preferentially expressed in a plant part, particularly the patatin gene from Solanum tuberosum, a 5'-untranslated leader, an open reading frame encoding a trehalose phosphate synthase activity, and downstream of said open reading frame a transcriptional terminator region.

According to yet another embodiment of the invention a chimaeric plant expressible gene is provided, comprising in sequence a transcription initiation region obtainable from a gene, preferentially expressed in a plant part, particularly the patatin gene from Solanum tuberosum, a 5'-untranslated leader, an open reading frame encoding a 4 trehalase coupled in the antisense orientation, and downstream of said open reading frame a transcriptional terminator region. A preferred plant expressible gene according to the invention is one wherein said transcriptional terminator region is obtainable from the proteinase inhibitor-II gene of Solanum tuberosum. The invention also provided vectors and recombinant plant genomes comprising a chimaeric plant expressible gene according to the invention, as well as a plant cell having a recombinant genome,- a plant or a part thereof, consisting essentially of cells. A further preferred plant species according to this aspect is Solanum tuberosum, and a micro-tuber thereof.

The invention further provides a process for obtaining trehalose, comprising the steps of growing plant cells according to the invention or cultivating a plant according S to the invention and extracting trehalose from said plant cells, plants or parts.

The following figures further illustrate the invention.

Throughout the description and claims of this specification the word "comprise" and variations of that word, such as "comprises" and "comprising", are not intended to exclude other additives or components or integers or steps.

DESCRIPTION OF THE FIGURES Figure 1. Schematic representation of binary vector pMOG845.

Figure 2. Schematic representation of multi-copy vector pMOG1192.

S 20 Figure 3. Alignments for maximal amino acid similarities of neutral trehalase from S. cerevisiae with periplasmatic trehalase from E. coli, small intestinal trehalase from rabbit and trehalase from pupal midgut of the silkworm, Bombyx mori. Identical residues among all trehalase enzymes are indicated in bold italics typeface.

Conserved regions of the amino acid sequences were aligned to give the best fit.

Gap's in the amino acid sequence are represented by dashes.

Positions of degenerated primers based on conserved amino acids are indicated by dashed arrows.

Figure 4. Alignment for maximal amino acid similarity of trehalases derived from E. coli (Ecoli2treh Ecolitreha), silkworm (Bommotreha), yellow mealworm (Tenmotreha), rabbit (Rabbitreha), Solanum tuberosum cv. Kardal (Potatotreha), and S. cerevisiae (Yeasttreha). Gap's in the amino C:WIWOaIRGAY NO"ELETE7l2 .DOC acid sequence are represented by dots.

Figure 5. Trehalase activity in leaf samples of Nicotiana tabacum cv.

Samsun NN. Non-transgenic control plants are indicated by letters a-1, plants transgenic for pMOG1078 are indicated by numbers.

Figure 6. Trehalose accumulation in microtubers induced on stem segments derived from Solanum tubersosum cv. Kardal plants transgenic for both pMOG 845 (patatin driven TPSE.coli expression) and pMOG1027 antisense-trehalase expression). N indicates the total number of transgenic lines screened. Experiments were performed in duplicate resulting in two values: a and b. ND: not determined.

DETAILED DESCRIPTION OF THE INVENTION 15 According to the present invention it has been found that the accumulation of an increased level of trehalose in plants and plant parts is feasible. This important finding can be exploited by adapting plant systems to produce and/or accumulate high levels of trehalose at lower cost.

20 According to one aspect of the invention the accumulation of increased levels of trehalose is achieved by inhibiting endogenous trehalases. Inhibition of trehalases can be performed basically in two ways: by administration of trehalase inhibitors exogenously, and by the production of trehalase inhibitors endogenously, for instance by transforming the plants with DNA sequences coding for trehalase inhibitors.

This inhibition can be equally well applied to plants which are transformed with enzymes which enable the production of trehalose, but also to plants which are able to synthesize trehalose naturally.

According to this first embodiment of the invention, trehalase inhibitors are administered to the plant system exogenously. Examples of trehalase inhibitors that may be used in such a process according to the invention are trehazolin produced in Micromonospora, strain SANK 62390 (Ando et al., 1991, J. Antibiot. 4A, 1165-1168), validoxylamine A, B, G, D-gluco-Dihydrovalidoxylamine A, L-ido-Dihydrovalidoxylamin

A,

Deoxynojirimycin (Kameda et al., 1987, J. Antibiot. 563-565), 6 epi-trehazolin (Trehalostatin) (Kobayashi Y. et al., 1994, J. Antiobiot.

Al, 932-938), castanospermin (Salleh H.M. Honek J.F. March 1990, FEBS 262(2), 359-362) and the 86kD protein from the american cockroach (Periplaneta americana) (Hayakawa et al., 1989, J. Biol. Chem. 2A6(27), 16165-16169).

A preferred trehalase inhibitor according to the invention is validamycin A 6-trideoxy-3-o-8-D-glucopyranosyl-5- (hydroxymethyl) trihydroxy-3- (hydroxymenthyl) -2-cyclohexen-l-yl] amino] -D-chiro-inositol) Trehalase inhibitors are administered to plants or plant parts, or plant cell cultures, in a form suitable for uptake by the plants, plant parts or cultures. Typically the trehalase inhibitor is in the form of an aqueous solution of between 100 nM and 10 mM of active ingredient, preferably between 0.1 and 1 mM. Aqueous solutions may be applied to plants or plant parts by spraying on leaves, watering, adding it to the 15 medium of a hydroculture, and the like. Another suitable formulation of validamycin is solacol, a commercially available agricultural formulation (Takeda Chem. Indust., Tokyo).

SAlternatively, or in addition to using exogenously administered trehalase inhibitors, trehalase inhibitors may be provided by introducing 20 the genetic information coding therefor. One form of such in-built trehalase inhibitor may consist of a genetic construct causing the production of RNA that is sufficiently complementary to endogenous RNA S.encoding for trehalase to interact with said endogenous transcript, thereby inhibiting the expression of said transcript. This so-called "antisense approach" is well known in the art (vide inter alia EP 0 240 208 A and the Examples to inhibit SPS disclosed in WO 95/01446).

A gene coding for trehalase has been isolated from a potato cDNA library and sequenced. The predicted amino acid sequence of trehalase as shown in SEQIDNO:10 is derived from the nucleotide sequence depicted in SEQIDNO: 9. A comparison of this sequence with known non-plant trehalase sequences learns that homology is scant. It is therefor questionable if such trehalase sequences used in an antisense approach are capable of inhibiting trehalase expression in planta.

Of course the most preferred embodiment of the invention is obtained by transforming a plant with the antisense trehalase gene which matches exactly with the endogenous trehalase gene. However, sequences 7 which have a high degree of homology can also be used. Thus, the antisense trehalase gene to be used for the transformation of potato will be directed against the nucleotide sequence depicted in SEQIDNO: 9.

It is also demonstrated in this application that the potato trehalase sequence can also be used to inhibit trehalase expression in tomato since the potato sequence is highly homologous to the tomato trehalase sequence. Thus,.it is envisaged that the potato sequence is usable at least in closely related species, but maybe also in other plants. This is even more the case, considering that it is usually enough to express only part of the homologous gene in the antisense orientation, in order to achieve effective inhibition of expression of the endogenous trehalase (vide Van der Krol et al., 1990, Plant Molecular Biology, 1A, 457-466).

Furthermore, it is shown in this application that the potato trehalase sequence can be used for the detection of homology in other species.

15 Trehalase gene sequences of other plants can be elucidated using several different strategies. One of the strategies is to use the isolated potato cDNA clone as a probe to screen a cDNA library containing the cDNA of the desired plant species. Positive reacting clones can then be isolated and subcloned into suitable vectors.

20 A second strategy to identify such genes is by purifying the oo proteins which are involved in trehalose degradation.

An example for such a strategy is the purification of a protein with acid invertase activity from potato (Solanum tuberosum tubers (Burch et al., Phytochemistry, Vol.31, No.6, pp. 1901-1904, 1992). The obtained protein preparation also exhibits trehalose hydrolysing activity.

Disaccharide hydrolysing activity of protein preparations obtained after purification steps can be monitored as described by Dahlqvist (Analytical Biochemistry 1, 18-25, 1964).

After purifying the protein(s) with trehalose hydrolysing activity to homogeneity, the N-terminal amino acid sequence or the sequence of internal fragments after protein digestion is determined. These sequences enable the design of oligonucleotide probes which are used in a polymerase chain reaction (PCR) or hybridization experiments to isolate the corresponding mRNAs using standard molecular cloning techniques.

Alternatively, degenerated primers can be designed based on conserved sequences present in trehalase genes isolated from other 8 species. These primers are used in a PCR strategy to amplify putative trehalase genes. Based on sequence information or Southern blotting, trehalase PCR fragments can be identified and the corresponding cDNA's isolated.

An isolated cDNA encoding a trehalose degrading enzyme is subsequently fused to a promoter sequence in such a way that transcription results in the synthesis of antisense mRNA.

Another form of such an in-built trehalase inhibitor may consist of a genetic construct causing the production of a protein that is able to inhibit trehalase activity in plants. A proteinaceous inhibitor of trehalase has been isolated and purified from the serum of resting adult american cockroaches (Periplaneta americana) (Hayakawa et al., supra) This protein, of which the sequence partly has been described in said publication, can be made expressable by isolation of the gene coding for 15 the protein, fusion of the gene to a suitable promoter, and transformation of said fused gene into the plant according to standard molecular biological methods.

A promoter may be selected from any gene capable of driving transcription in plant cells.

S 20 If trehalose accumulation is only desired in certain plant parts, such as potato (mini-)tubers, the trehalase inhibitory DNA construct the antisense construct) comprises a promoter fragment that is preferentially expressed in (mini-)tubers, allowing endogenous trehalase levels in the remainder of the plant's cells to be substantially unaffected. Thus, any negative effects of trehalose to neighbouring plant cells due to trehalose diffusion, is counteracted by unaffected endogenous trehalase activity in the remainder of the plant.

In the Example illustrating the invention, wherein trehalose phosphate synthase is produced under the control of the patatin promoter fragment, also the trehalase-inhibitory construct may comprise a promoter fragment of the patatin gene.

Mutatis mutandis if trehalose is to be accumulated in tomato fruit, both a plant expressible trehalose phosphate synthase gene, which is at least expressed in the tomato fruit is to be used, as well as a plant expressible trehalase-inhibitory DNA construct, which should be expressed preferentially in the fruit, and preferably not, or not substantially, 9 outside the fruit. An example of a promoter fragment that may be used to drive expression of DNA-constructs preferentially in tomato fruit is disclosed in EP 0 409 629 Al. Numerous modifications of this aspect of the invention, that do not depart from the scope of this invention, are readily envisaged by persons having ordinary skill in the art to which this invention pertains.

An alternative method to block the synthesis of undesired enzymatic activity such as caused by endogenous trehalase is the introduction into the genome of the plant host of an additional copy of said endogenous trehalase gene. It is often observed that the presence of a transgene copy of an endogenous gene silences the expression of both the endogenous gene and the transgene (EP 0 465 572 Al).

According to one embodiment of the invention accumulation of trehalose is brought about in plants wherein the capacity of producing 15 trehalose has been introduced by introduction of a plant expressible gene construct encoding trehalose phosphate synthase (TPS), see for instance WO 95/06126.

Any trehalose phosphate synthase gene under the control of regulatory elements necessary for expression of DNA in plant cells, either specifically or constitutively, may be used, as long as it is 0 capable of producing active trehalose phosphate synthase activity. Most preferred are the trehalose phosphate synthase genes which also harbour a coding sequence for trehalose phosphate phosphatase activity, the so called bipartite enzymes. Such a gene, formerly only known to exist in yeast (see e.g. WO 93/17093), can also been found in most plants. This 00* application describes the elucidation of such a gene from the sunflower Helianthus annuus, while also evidence is given for the existence of a homologous gene in Nicotiana tabacum. It is believed that the use of a bipartite enzyme enhances the production of trehalose because it enables completion of the metabolic pathway from UDP-glucose and glucose-6phosphate into trehalose at one and the same site. Hence, the two-step synthesis is simplified into a one-step reaction, thereby increasing reaction speed and, subsequently, trehalose yield.

As genes involved in trehalose synthesis, especially genes coding for bipartite enzymes, become available from other sources these can be used in a similar way to obtain a plant expressible trehalose synthesizing gene according to the invention.

Sources for isolating trehalose synthesizing activities include microorganisms bacteria, yeast, fungi), but these genes can also be found in plants and animals.

The invention also encompasses nucleic acid sequences which have been obtained by modifying the nucleic acid sequence encoding enzymes active in the synthesis of trehalose by mutating one or more codons so that it results in amino acid changes in the encoded protein, as long as mutation of the amino acid sequence does not entirely abolish trehalose synthesizing activity.

According to another embodiment of the invention, plants are genetically altered to produce and accumulate trehalose in specific parts of the plant, which were selected on the basis of considerations such as substrate availability for the enzyme, insensitivity of the plant part to 15 any putative adverse effects of trehalose on plant cell functioning, and the like. A preferred site for trehalose synthesising enzyme expression are starch storage parts of plants. In particular potato tubers are considered to be suitable plant parts. A preferred promoter to achieve selective enzyme expression in microtubers and tubers of potato is 20 obtainable from the region upstream of the open reading frame of the patatin gene of potato (Solanum tuberosum) Plants provide with a gene coding for trehalose phosphate synthase only may be further modified by introducing additional genes that encode 0* phosphatases that are capable of the conversion of trehalose phosphate into trehalose. At least in potato tubers or micro-tubers, potato leaves and tobacco leaves and roots, endogenous phosphatase activity appears to be present, so that the introduction of a trehalose phosphate phosphatase (TPP) gene is not an absolute requirement.

Preferred plant hosts among the Spermatophyta are the Angiospermae, notably the Dicotyledoneae, comprising inter alia the Solanaceae as a representative family, and the Monocotyledoneae, comprising inter alia the Gramineae as a representative family. Suitable host plants, as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant DNA techniques to cause or enhance production of trehalose in the desired plant or plant organ; these plants 11 may be used directly the plant species which produce edible parts) in processing or the trehalose may be extracted and/or purified from said host. Crops with edible parts according to the invention include those which have flowers such as cauliflower (Brassica oleracea), artichoke (Cynara scolymus), fruits such as apple (Malus, e.g. domesticus), banana (Musa, e.g. acuminata), berries (such as the currant, Ribes, e.g.

rubrum), cherries (such as the sweet cherry, Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus), grape (Vitis, e.g. vinifera), lemon (Citrus limon), melon (Cucumis melo), nuts (such as the walnut, Juglans, e.g. regia; peanut, Arachis hypogeae), orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica), pear (Pyra, e.g. communis), pepper (Solanum, e.g. capsicum), plum (Prunus, e.g. domestica), strawberry (Fragaria, e.g. moschata), tomato (Lycopersicon, e.g. esculentum), leafs, such as alfalfa (Medicago sativa), cabbages (such as Brassica oleracea), S* 15 endive (Cichoreum, e.g. endivia), leek (Allium porrum), lettuce (Lactuca sativa), spinach (Spinaciaoleraceae), tobacco (Nicotiana tabacum), roots, such as arrowroot (Maranta arundinacea), beet (Beta vulgaris), carrot (Daucus carota), cassava (Manihot esculenta), turnip (Brassica rapa), radish (Raphanus sativus), yam (Dioscorea esculenta), sweet potato 20 (Ipomoea batatas) and seeds, such as bean (Phaseolus vulgaris), pea (Pisum sativum), soybean (Glycin max), wheat (Triticum aestivum), barley (Hordeum vulgare), corn (Zea mays), rice (Oryza sativa), tubers, such as kohlrabi (Brassica oleraceae), potato (Solanum tuberosum), and the like.

The edible parts may be conserved by drying in the presence of enhanced trehalose levels produced therein due to the presence of a plant expressible trehalose phosphate synthase gene.

The method of introducing the plant expressible gene coding for a trehalose-synthesizing enzyme, or any other sense or antisense gene into a recipient plant cell is not crucial, as long as the gene is expressed in said plant cell. The use of Agrobacterium tumefaciens or Agrobacterium rhizogenes mediated transformation is preferred, but other procedures are available for the introduction of DNA into plant cells. Examples are transformation of protoplasts using the calcium/polyethylene glycol method, electroporation, microinjection and DNA-coated particle bombardment (Potrykus, 1990, Bio/Technol. 1, 535-542). Also combinations of Agrobacterium and coated particle bombardment may be used. Also 12 transformation protocols involving other living vectors than Agrobacterium may be used, such as viral vectors from the Cauliflower Mosaic Virus (CaMV) and or combinations of Agrobacterium and viral vectors, a procedure referred to as agroinfection (Grimsley N. et al., 8 January 1987, Nature 325, 177-179). After selection and/or screening, the protoplasts, cells or plant parts that have been transformed are .regenerated into whole plants, using methods known in the art (Horsch et al., 1985, Science 22., 1229-1231) The development of reproducible tissue culture systems for monocotyledonous crops, together with methods for introduction of genetic material into plant cells has facilitated transformation. Presently, preferred methods for transformation of monocot species are transformation with supervirulent Agrobacterium-strains, microprojectile bombardment of explants or suspension cells, and direct DNA uptake or 15 electroporation (Shimamoto, et al., 1989, Nature 33, 274-276).

Agrobacterium-mediated transformation is functioning very well in rice (WO 94/00977). Transgenic maize plants have been obtained by introducing the Streptomnyces hygroscopicus bar-gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin), into embryogenic cells of a maize suspension culture g 'by microprojectile bombardment (Gordon-Kamm, 1990, Plant Cell, 2, 603-618). The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee, 1989, Plant Mol. Biol. 21-30). Wheat plants have been regenerated from embryogenic suspension culture by selecting only the aged compact and nodular embryogenic callus tissues for the establishment of the embryogenic suspension cultures (Vasil, 1990 Bio/Technol. a, 429-434).

Suitable DNA sequences for control of expression of the plant expressible genes (including marker genes), such as transcriptional initiation regions, enhancers, non-transcribed leaders and the like, may be derived from any gene that is expressed in a plant cell. Also intended are hybrid promoters combining functional portions of various promoters, or synthetic equivalents thereof. Apart from constitutive promoters, inducible promoters, or promoters otherwise regulated in their expression pattern, e.g. developmentally or cell-type specific, may be used to control expression of the plant expressible genes according to the 13 invention as long as they are expressed in plant parts that contain substrate for TPS.

To select or screen for transformed cells, it is preferred to include a marker gene linked to the plant expressible gene according to .the invention to be transferred to a plant cell. The choice of a suitable marker gene in plant transformation is well within the scope .of the average skilled .worker; some examples of routinely used marker genes are the neomycin phosphotransferase genes conferring resistance to kanamycin (EP-B 131 623), the glutathion-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides (EP-A 256 223), glutamine synthetase conferring upon overexpression resistance to glutamine synthetase inhibitors such as phosphinothricin (W087/05327), the acetyl transferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin (EP-A 275 S 15 957), the gene encoding a 5-enolshikimate-3- phosphate synthase (EPSPS) conferring tolerance to N-phosphonomethylglycine, the bar gene conferring resistance against Bialaphos WO 91/02071) and the like. The actual choice of the marker is not crucial as long as it is functional (i.e.

selective) in combination with the plant cells of choice.

The marker gene and the gene of interest do not have to be linked, since co-transformation of unlinked genes Patent 4,399,216) is also an efficient process in plant transformation.

Preferred plant material for transformation, especially for dicotyledonous crops are leaf-discs which can be readily transformed and have good regenerative capability (Horsch R.B. et al., (1985) Science 221, 1229-1231).

It is immaterial to the invention how the presence of two or more genes in the same plant is effected. This can inter alia done be achieved by one of the following methods: transformation of the plant line with a multigene construct containing more than one gene to be introduced, co-transforming different constructs to the same plant line simultaneously, subsequent rounds of transformation of the same plant with the genes to be introduced, crossing two plants each of which contains a different gene to be introduced into the same plant, or combinations thereof.

The field of application of the invention lies both in agriculture and horticulture, for instance due to improved properties of the modified plants as such stress tolerance, such as cold tolerance, and preferably drought resistance, and increase in post-harvest quality and shelf-life of plants and plant products), as well as in any form of industry where trehalose is or will be applied in a process of forced water extraction, such as drying or freeze drying. Trehalose can be used or sold as such, for instance in purified form or in admixtures, or in the form of a plant product, such as a tuber, a fruit, a flower containing the trehalose, either in native state or in (partially) dehydrated form, and the like. Plant parts harbouring (increased levels of) trehalose phosphate or trehalose may be used or sold as such or 15 processed without the need to add trehalose.

Also trehalose can be extracted and/or purified from the plants or plant parts producing it and subsequently used in an industrial process.

In the food industries trehalose can be employed by adding trehalose to foods before drying. Drying of foods is an important method of 20 preservation. Trehalose seems especially useful to conserve food products through conventional air-drying, and to allow for fast reconstitution upon addition of water of a high quality product (Roser et al., July 1991, Trends in Food Science and Technology, pp. 166-169). The benefits include retention of natural flavors/fragrances, taste of fresh product, and nutritional value (proteins and vitamins). It has been shown that trehalose has the ability to stabilize proteins e.g. vaccines, enzymes and membranes, and to form a chemically inert, stable glass. The low water activity of such thoroughly dried food products prevents chemical reactions, that could cause spoilage.

Field crops like corn, cassava, potato, sugar beet and sugarcane have since long been used as a natural source for bulk carbohydrate production (starches and sucrose). The production of trehalose in such crops, facilitated by genetic engineering of the trehalose-biosynthetic pathway into these plant species, would allow the exploitation of such engineered crops for trehalose production.

Trehalose is also used in drying or storage of biological macromolecules, such as peptides, enzymes, polynucleotides and the like.

All references cited in this specification are indicative of the level of skill in the art to which the invention pertains. All publications, whether patents or otherwise, referred to previously or later in this specification are herein incorporated by reference as if each of them was individually incorporated by reference. In particular WO 95/01446, cited herein, describing the production of trehalose in higher plants by genetic manipulation is herein incorporated by reference.

The Examples given below illustrate the invention and are in no way intended to indicate the limits of the scope of the invention.

Experimental DNA manipulations All DNA procedures (DNA isolation from E.coli, restriction, ligation, transformation, etc.) are performed according to standard protocols (Sambrook et al. (1989) Molecular Cloning: a laboratory manual, 2nd ed.

Cold Spring Harbor Laboratory Press, CSH, New York).

Strains oo In all examples E.coli K-12 strain DH5( is used for cloning. The Agrobacterium tumefaciens strains used for plant transformation experiments are EHA 105 and MOG 101 (Hood et al. 1993, Trans. Research 2, *208-218) Isolation of a patatin promoter/construction of pMOG546 A patatin promoter fragment is isolated from chromosomal DNA of Solanum tuberosum cv. Bintje using the polymerase chain reaction. A set of oligonucleotides, complementary to the sequence of the upstream region of the Apat21 patatin gene (Bevan, Barker, Goldsbrough, Jarvis, Kavanagh, T. and Iturriaga, G. (1986) Nucleic Acids Res. J1: 5564- 5566), is synthesized consisting of the following sequences: AAG CTT ATG TTG CCA TAT AGA GTA G 3' PatB33.2 (SEQIDNO:3) 5' GTA GTT GCC ATG GTG CAA ATG TTC 3' PatATG.2 (SEQIDNO:4) 16 These primers are used to PCR amplify a DNA fragment of 1123bp, using chromosomal DNA isolated from potato cv. Bintje as a template. The amplified fragment shows a high degree of similarity to the Xpat21 patatin sequence and is cloned using EcoRI linkers into a pUC18 vector resulting in plasmid pMOG546.

Construction of pMOG 799 pMOG 799 harbours the TPS gene from E. coli under control of the double enhanced 35S Cauliflower Mosaic promoter. The construction of this binary vector is described in detail in International patent application WO 95/01446, incorporated herein by reference.

Construction of DMOG845.

Plasmid pMOG546 containing the patatin promoter is digested with NcoI- 15 KpnI, incubated with E. coli DNA polymerase I in the presence of dATP and e dCTP thereby destroying the NcoI and KpnI site and subsequently relegated. From the resulting vector a l.lkb EcoRI-Smal fragment containing the patatin promoter is isolated and cloned into pMOG798 (described in detail in WO 95/01446) linearized with SmaI-EcoRI consequently exchanging the 35S CaMV promoter for the patatin promoter.

The resulting vector is linearized with HindIII and ligated with the following oligonucleotide duplex: (HindIII) PstI KpnI HindIII 5' AGCT CTGCAG TGA GGTACC A 3' TCV 11 3' GACGTC ACT CCATGG TTCGA 5' TCV 12 (SEQIDNO:6) After checking the orientation of the introduced oligonucleotide duplex, the resulting vector is linearized with PstI-HindIII followed by the insertion of a 950bp PstI-HindIII fragment harbouring the potato proteinase inhibitor II terminator (PotPiII) (An, Mitra, Choi, Costa, An, Thornburg, R. W. and Ryan, C.A. (1989) The Plant Cell 1: 115-122 The PotPiII terminator is isolated by PCR amplification using chromosomal DNA isolated from potato cv. Desiree as a template and the following set of oligonucleotides: 17 GTACCCTGCAGTGTGACCCTAGAC 3' TCV 15 (SEQIDNO:7) TCGATTCATAGAAGCTTAGAT 3' TCV 16 (SEQIDNO:8) The TPS expression cassette is subsequently cloned as a EcoRI-HindIII fragment into the binary vector pMOG402 resulting in pMOG845 (fig. 1).

A sample of E.coli Dha strain, harbouring pMOG845 has been deposited at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, P.O. Box 273, 3740 AG Baarn, The Netherlands, on January 4, 1995; the Accession Number given by the International Depositary Institution is CBS 101.95.

Triparental matings The binary vectors are mobilized in triparental matings with the E. coli strain HB101 containing plasmid pRK2013 (Ditta Stanfield, Corbin, 15 and Helinski, D.R. et al. (1980) Proc. Natl. Acad. Sci. USA 11, 7347) into Agrobacterium tumefaciens strain MOG101 or EHA105 and used for transformation.

Transformation of tobacco (Nicotiana tabacum SR1I Tobacco is transformed by cocultivation of plant tissue with Agrobacteriumtumefaciens strain MOG101 containing the binary vector of interest as described. Transformation is carried out using cocultivation of tobacco (Nicotiana tabacum SR1) leaf disks as described by Horsch et 'al. 1985, Science 227, 1229-1231. Transgenic plants are regenerated from 25 shoots that grow on selection medium containing kanamycin, rooted and transferred to soil.

*4 Transformation of potato tuber discs Potato (Solanum tuberosum cv. Kardal) is transformed with the Agrobacterium strain EHA 105 containing the binary vector of interest.

The basic culture medium is MS30R3 medium consisting of MS salts (Murashige, T. and Skoog, F. (1962) Physiol. Plan. 1i, 473), R3 vitamins (Ooms et al. (1987) Theor. Appl. Genet. 744), 30 g/l sucrose, 0.5 g/l MES with final pH 5.8 (adjusted with KOH) solidified when necessary with 8 g/l Daichin agar. Tubers of Solanum tuberosum cv. Kardal are peeled and surface sterilized by burning them in 96% ethanol for 5 seconds.

Extinguish the flames in sterile water and cut slices of approximately 2 mm thickness. Disks are cut with a bore from the vascular tissue and incubated for 20 minutes in MS30R3 medium containing 1-5 xl08 bacteria/mi of Agrobacterium EHA 105 containing the binary vector. Wash the tuber discs with MS30R3 medium and transfer them to solidified postculture medium PM consists of M30R3 medium supplemented with 3.5 mg/l zeatin riboside and 0.03 mg/l indole acetic acid (IAA). After two days, discs were transferred to fresh PM medium with 200 mg/l cefotaxim and 100 mg/l vancomycin. Three days later, the tuber discs are transferred to shoot induction medium (SIM) which consists of PM medium with 250 mg/l carbenicillin and 100 mg/l kanamycin. After 4-8 weeks, shoots emerging from the discs are excised and placed on rooting medium (MS30R3-medium with 100 mg/l cefotaxim, 50 mg/l vancomycin and 50 mg/l kanamycin). The shoots are propagated axenically by meristem cuttings.

o Potato stem-segment transformation protocol.

Potato transformation experiments using stem-internodes were performed in a similar way as described by Newell C.A. et al., Plant Cell Reports 30-34, 1990.

Induction of micro-tubers r Stem segments of in vitro potato plants harbouring an auxiliary meristem are transferred to micro-tuber inducing medium. Micro-tuber inducing medium contains 1 X MS-salts supplemented with R3 vitamins, 0.5 g/l MES 25 (final pH= 5.8, adjusted with KOH) and solidified with 8 g/1 Daishin agar, 60 g/l sucrose and 2.5 mg/1 kinetin. After 3 to 5 weeks of growth in the dark at 24 0 C, micro-tubers are formed.

Trehalose assay Trehalose was determined quantitatively by anion exchange chromatography with pulsed amperometric detection. Extracts were prepared by adding 1 ml boiling water to 1 g frozen material which was subsequently heated for at 100 0 C. Samples (25 p.l) were analyzed on a Dionex DX-300 liquid chromatograph equipped with a 4 x 250 mm Dionex 35391 carbopac PA-1 column and a 4 x 50 mm Dionex 43096 carbopac PA-1 precolumn. Elution was with 100 mM NaOH at 1 ml/min. Sugars were detected with a pulsed 19 amperometric detector (Dionex, PAD-2). Commercially available trehalose (Sigma) was used as a standard.

Isolation of Validamycin A Validamycin A is isolated from Solacol, a commercial agricultural formulation (Takeda Chem. Indust., Tokyo) as described by Kendall et al.

(1990) Phytochemistry, Vol. 21, No. 8, pp. 2525-2528. The procedure involves ion exchange chromatography (QAE-Sephadex A-25 (Pharmacia), bed vol. 10 ml, equilibration buffer 0.2 mM Na-Pi pH 7) from a 3% agricultural formulation of Solacol. Loading 1 ml of Solacol on the column and eluting with water in 7 fractions, practically all Validamycin is recovered in fraction 4.

Based on a 100% recovery, using this procedure, the concentration of Validamycin A was adjusted to 110-3 M in MS-buffer, for use in trehalose accumulation tests.

Alternatively, Validamycin A and B may be purified directly from Streptomyces hygroscopicus var. limoneus, as described by Iwasa T. et al., 1971, in The Journal of Antibiotics ZA(2), 119-123, the content of 20 which is incorporated herein by reference.

Construction of pMOG1027 pMOG1027 harbours the trehalase gene from Solanum tuberosum cv. Kardal in the reversed orientation under control of the double enhanced 25 Cauliflower Mosaic promoter. The construction of this vector is very similar to the construction of pMOG799 and can be performed by any person skilled in the art. After mobilization of this binary vector by triparental mating to Agrobacterium, this strain can be used to transform plant cells and to generate transgenic plants having reduced levels of trehalase activity.

Construction of pMOG1028 pMOG1028 harbours the trehalase gene from Solanum tuberosum cv. Kardal in the reversed orientation under control of the tuber specific patatin promoter. The construction of this vector is very similar to the construction of pMOG845 and can be performed by any person skilled in the art. After mobilization of this binary vector by triparental mating to Agrobacterium, this strain can be used in potato transformation experiments to generate transgenic plants having reduced levels of trehalase activity in tuber-tissue.

Construction of DMOG 1078 To facilitate the construction of a binary expression cassette harbouring the trehalase cDNA clone in the "sense" orientation under control of the double enhanced 35S CaMV promoter, two HindIII sites were removed from the trehalase cDNA coding region (without changing the amino acid sequence) by PCR based point-mutations. In this way, a BamHI fragment was engineered that contained the complete trehalase open reading frame. This fragment was subsequently used for cloning in the binary vector pMOGB00 behind the constitutive de35S CaMV promoter yielding pMOG1078. pMOG800 is derived from pMOG402; the KpnI site in the polylinker has been restored.

pMOG402 is derived of pMOG23 (described in WO 95/01446) and harbours a restored neomycin phosphotransferase gene (Yenofsky Fine Pellow Proc Natl Acad Sci USA 87: 3435-3439, 1990) o 20 EXAMPLE 1 Trehalose production in tobacco plants transformed with pMOG799 Tobacco leaf discs are transformed with the binary vector pMOG799 using Agrobacterium tumefaciens. Transgenic shoots are selected on kanamycin.

Transgenic plants are transferred to the greenhouse to flower and set 25 seed after selfing (Sl) Seeds of these transgenic plants are surface sterilised and germinated in vitro on medium with Kanamycin. Kanamycin resistant seedlings and wild-type tobacco plants are transferred to MSmedium supplemented with 10-3 M validamycin A. As a control, transgenic seedlings and wild-type plants are transferred to medium without Validamycin A. Analysis of leaves and roots of plants grown on Validamycin A shows elevated levels of trehalose compared to the control plants (Table No trehalose was detected in wild-type tobacco plants.

Table 1 pMOG799.1 pMOG799.13 pMOG799.31 Wild-type SR1 with Validamycin A leaf roots 0.0081 0.0044 0.0110 0.0080 0.0008 0.0088 without Validamycin A leaf roots 0.003 EXAMPLE 2 Trehalose production in potato micro-tubers transformed with pMOG845 Potato Solanum tuberosum cv. Kardal tuber discs are transformed with Agrobacterium tumefaciens EHA105 harbouring the binary vector pMOG845.

Transgenic shoots are selected on kanamycin. Micro-tubers (m-tubers) are induced on stem segments of transgenic and wild-type plants cultured on m-tuber inducing medium supplemented with 10-3 M Validamycin A. As a control, m-tubers are induced on medium without Validamycin A. M-tubers induced on medium with Validamycin A showed elevated levels of trehalose in comparison with m-tubers grown on medium without Validamycin A (Table No trehalose was detected in wild-type m-tubers.

Table 2.

r Trehalose fresh weight) +Validamycin A -Validamycin A 0.016 845-2 845-4 845-8 845-13 845-22 845-25 wT Kardal 0.051 0.005 0.121 0.002 EXAMPLE 3 Trehalose production in hydrocultures of tobacco plants transformed with pMOG799 Seeds (Sl) of selfed tobacco plants transformed with the binary vector pMOG799 are surface sterilised and germinated in vitro on MS20MS medium containing 50 gg/ml Kanamycin. Kanamycin resistant seedlings are transferred to soil and grown in a growth chamber (temp. 23 0 C, 16 hours of light/day). After four weeks, seedlings were transferred to hydrocultures with ASEF clay beads with approximately 450 ml of medium.

The medium contains 40 g/l Solacol dissolved in nano-water buffered with g/l MES to adjust to pH 6.0 which is sieved through a filter to remove solid particles. Essential salts are supplemented by adding POKONTM (1.5 ml/1). The following antibiotics are added to prevent growth of micro-organisms: 500g/ml Carbenicillin, 40gg/ml Nystatin and 100g/ml Vancomycin. As a control, transgenic seedlings and wild-type plants are transferred to medium without Solacol. Analysis of leaves of plants grown on Solacol shows elevated levels of trehalose compared to the control plants (Table No trehalose was detected in wild-type tobacco plants.

Table 3 Solacol Trehalose pMOG 799.1-1 0.008 pMOG 799.1-2 0.004 pMOG 799.1-3 20 pMOG 799.1-4 pMOG 799.1-5 0.008 pMOG 799.1-6 S- pMOG 799.1-7 0.005 pMOG 799.1-8 25 pMOG 799.1-9 pMOG 799.1-10 0.007 Wild-type SR1-1 Wild-type SR1-2 Wild-type SR1-3 Wild-type SR1-4 Example 4 Cloning of a full lenrth cDNA encoding trehalase from potato tuber Using the amino acid sequence of the conserved regions of known trehalase genes (E.coli, Yeast, Rabbit, B. mori) (fig. four degenerated primers were designed: C CC CGT GT A TTAT GG GGI G TT IGA T TA TGGGAC Tase24 (SEQIDNO:11) T A A TAA AG C CGGC TAA GT GTICCIGGIGGICGITT IGA T Tase25 (SEQIDNO:12) CGT AG T GA TG A A GGIGG TGI ICGI IAG TA TA Tase26 (SEQIDNO:13) C CT CA G G C G AT A I C TTI CCATCC AAICCITC Tase27 (SEQIDNO:14) GA GC G Combinations of these primers in PCR experiments with genomic DNA and cDNA from S. tuberosum cv. Kardal leaf and tuber material respectively as template, resulted in several fragments of the expected length. A number of 190 bp. fragments obtained with the primer combination Tase24 and Tase 26 were subcloned into a pGEM T vector and sequenced. Several of the clones analyzed showed homology with known trehalase sequences.

To exclude the isolation of non-plant derived trehalase sequences, Southern blot analysis was performed with gDNA from potato cv. Kardal. A number of clones isolated did not cross-hybridize with Kardal genomic DNA and were discarded. Two isolated clones were identical, gTasel5.4 derived 35 from a genomic PCR experiment and cTase5.2 derived from a PCR on cDNA, both showing hybridization in Southern blot analysis. One single hybridizing band was detected (EcoRI 1.5 Kb, HindIII 3 Kb and BamHI larger than 12 Kb) suggesting the presence of only one copy of the isolated PCR fragment.

A cDNA library was constructed out of poly A RNA from potato tubers (cv.

Kardal) using a Stratagene cDNA synthesis kit and the vector Lambda ZAPII. Recombinant phages (500.000) were screened with the radiolabeled cTase5.2 PCR fragment resulting in the identification of 3 positive clones. After purification, two clones were characterised with restriction enzymes revealing inserts of 2.15 and 2.3 kb respectively.

Their nucleotide sequence was 100% identical. The nucleic acid sequence of one of these trehalase cDNA clones from Solanum tuberosum including 24 its open reading frame is depicted in SEQIDNO:9, while the aminoacid sequence derived from this nucleic acid sequence is shown in A plasmid harbouring an insert comprising the genetic information coding for trehalase has been deposited under no. CBS 804.95 with the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, P.O. Box 273, 3740 AG Baarn, the Netherlands on December 8, 1995.

EXAMPLE Homology between the trehalase gene from potato with other Solanaceae Genomic DNA was isolated from tomato (Lycopersicon esculentum cv. Money maker), tobacco (Nicotiana tabacum cv. Petit havanna, SR1) and potato (Solanum tuberosum cv. Kardal), and subsequently digested with the restriction enzymes BamHI, BglII, NcoI, Spel, AccI, HindIII and EcoRI.

After gel-electrophoresis and Southern blotting, a 3 2 p]-alpha dCTP labelled trehalase potato cDNA probe was hybridized to the blot.

Hybridization signals of almost similar strength were observed in the lanes with potato and tomato genomic DNA indicating a high degree of identity. Only a weak hybridization signal was observed in the lanes harbouring tobacco genomic DNA indicating a low degree of identity. A similar strategy can be used to identify trehalase genes from other crops and to select for crops were trehalase activity can be eliminated, via the anti-sense expression strategy, using a heterologous trehalase cDNA clone with sufficient homology. Alternatively, a homologous trehalase cDNA clone can be isolated and used in the anti-sense expression strategy.

EXAMPLE 6 Overexression of a potato trehalase cDNA in Nicotiana tabacum Tobacco leaf discs are transformed with the binary vector pMOG1078 using Agrobacterium tumefaciens. Transgenic shoots are selected on kanamycin and transferred to the greenhouse. Trehalase activity was determined in leaf samples of 26 transgenic and 12 non-transgenic control plants (Fig.

Trehalase activity up to ca. 17 g trehalose/h/ig protein was measured compared to ca. 1 pg trehalose/h/g protein for non-transgenic controls. This clearly confirms the identity of the potato trehalase cDNA.

EXAMPLE 7 Transformation of pMOG845 transgenic potato plants with DMOG1027 In order to super-transform pMOG845 transgenic potato lines with an antisense trehalase construct (pMOG1027), stem segments were cut from in vitro cultured potato shoots transgenic for pMOG845. Three parent lines were selected, pMOG845/11, /22 and /28 that revealed to accumulate trehalose in microtubers when grown on validamycin A. The stem segments were transformed with the binary vector pMOG1027 using Agrobacterium tumefaciens. Supertransformants were selected on Hygromycin and grown in vitro.

EXAMPLE 8 Trehalose production in tubers of potato plants transgenic for DMOG845 and pMOG1027 Microtubers were induced on explants of the pMOG845 transgenic potato S.1 plants supertransformed with pMOG1027 using medium without the trehalase inhibitor validamycin A. The accumulation of trehalose, up to 0.75 mg.g-1 fresh weight, was noted in the supertransformed lines proving the reduced 20 trehalase activity in these lines using the anti-sense trehalase expression strategy (Fig. 6).

EXAMPLE 9 Isolation of a bipartite TPS/TPP gene from Helianthus annuus 25 To isolate a bipartite clone from H. annuus, a PCR amplification experiment was set up using two degenerate primers, TPS-deg2 and TPS- This primerset was used in combination with cDNA constructed on H.

annuus leaf RNA as a template. A DNA fragment of approximately 650 bp.

was amplified having a high similarity on amino acid level when compared to tps coding regions from E. coli and yeast. Based on its nucleotide sequence, homologous primers were designed and used in a Marathon RACE protocol (Clontech) to isolate the 5' and 3' parts of corresponding tps cDNA's. Using primercombinations SUNGSP1 (or 2)/AP1 in RACE PCR, no bands were observed whereas nested PCR with NSUNGSP1(or2)/AP2 resulted in several DNA fragments. Some of these fragments hybridized with a 32P labelled Sunflower tps fragment after Southern blotting. Two fragments of circa 1.2 kb and 1.7 kb, corresponding respectively to the 5' and 3' part, were isolated from gel, subcloned and sequenced. The nucleotide sequence revealed a clear homology with known tps and tpp sequences indicating the bipartite nature of the isolated cDNA (SEQ ID NO Using a unique XmaI site present in both fragments, a complete TPS/TPP bipartite coding region was obtained and subcloned in pGEM-T (Promega) yielding pMOG1192 (Fig. 2).

TPSdeg2: tig git kit tyy tic aya yic cit tyc c (SEQIDNO: 23) TPSdeg5: gyi aci arr ttc ati ccr tci c (SEQIDNO: 27) SUNGSP1: cga aac ggg ccc atc aat ta (SEQIDNO: SUNGSP2: tcg atg aga tca atg ccg ag (SEQIDNO: 16) AP1 (Clontech): cca tcc taa tac gac tca cta tag ggc (SEQIDNO: 17) NSUNGSP1: cac aac agg ctg gta tcc cg (SEQIDNO: 18) NSUNGSP2: caa taa cga act ggg aag cc (SEQIDNO: 19) AP2 (Clontech): act cac tat agg gct cga gcg gc (SEQIDNO: FEXAMPLE 20 Isolation of a bipartite TPS/TPP gene from Nictiana tabacum SAnother strategy to isolate bipartite TPS/TPP genes from plants or other organisms involved the combined use of TPS and TPP primers in a single PCR reaction. As an example, a PCR was performed using cDNA generated on 25 tobacco leaf total RNA and the primerset TPSdegl and TRE-TPP-16. Nested PCR, using the amplification mix of the first reaction as template, with TPSdeg2 and TRE-TPP-15 resulted in a DNA fragment of ca. 1.5 kb. Nested PCR of the original amplification mix with TPSdeg2 and TRE-TPP-10 yielded a DNA fragment of ca.1.

2 kb.

Initial amplification using primer combination TPSdegl and TRE-TPP-6 followed by a nested PCR using primer combination TPSdeg2 and yielded a DNA fragment of ca. 1.5 kb.

Based on sequence analysis, the 1.2 kb and 1.5 kb amplified DNA fragments displayed a high degree of identity to TPS and TPP coding regions indicating that they encode a bipartite TPS/TPP proteins.

TPSdegl: TRE-TPP-16: TPSdeg2: TRE-TPP-10: TRE-TPP-6: GAY ITI ATI CCI ACI TIG GIT TGR TCI CCR TGY TCR TCI TGG RTI CAI GCR TYY TIC CAY GAY TAY CA AAI AC AYA YIC CIT TYC C TTI C

(SEQIDNO:

(SEQIDNO:

(SEQIDNO:

(SEQIDNO:

(SEQIDNO:

(SEQIDNO:

21) 22) 23) 24) 26) ARI ARY TCY TCI GCI SWI ARI CC GTR AAR TCR TCI CC 9 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: MOGEN INTERNATIONAL NV STREET: Einsteinweg 97 CITY: Leiden COUNTRY: The Netherlands POSTAL CODE (ZIP): 2233 CB TELEPHONE: (31) 71-5258282 TELEFAX: (31) 71-5221471 (ii) TITLE OF INVENTION: Enhanced accumulation of trehalose in plants (iii) NUMBER OF SEQUENCES: 27 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 2621 base pairs 30 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO S(ix) FEATURE: NAME/KEY: CDS LOCATION: 25..2485 OTHER INFORMATION: /function= "trehalose phosph.

synthase and trehalose phosph. phosphatase" /product= "bipartite enzyme" (ix) FEATURE: NAME/KEY: unsure LOCATION: 1609..1611 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTGATCCTGC GGTTTCATCA. CAAT ATG ATA CTC TTA CAT CTG ATG CCC CTT Met Ile Leu Leu His Leu Met Pro Leu 1 CAG ATG CTC CCA AAT AGG TTG ATT GTC GTA TCG AAT CAG TTA CCC ATA Gin Met Leu Pro Asn Arg Leu Ile Val Val Ser Asn-Gin ATC GCT AGG CTA AGA Ile Ala Arg Leu Arg CTA ACG ACA ATG GAG GGT CCT TTT Leu Thr'Thr Met Giu Giy Pro Phe 35 Leu Pro Ile GGG ATT TCA Gly Ile Ser CAT TAC CCG His Tyr Pro GCT GAC GTT Aia Asp Val CTT GGG ACG Leu Giy Thr CAG CCG TTG Gin Pro Leu GTT CGA TTT ACA Val Arg Phe Thr ACA TCA AAG ATG Thr Ser Lys Met AGG TTT TCT ATT Arg Phe Ser Ile GGC GAT CCA CTA Giy Asp Pro Leu foe GOC CCT Giy Pro ACC GAA CAA GAT Thr Giu Gin Asp GTG TCA AAG ACA Val Ser Lys Thr CTC GAT AGG TTT Leu Asp Arg Phe

AAT

Asn TGC GTT GCG GTT Cys Vai Ala Val

TTT

Phe 95 GTC CCT ACT TCA Val Pro Thr Ser

AAA

Lys 100 TGG GAC CAA TAT Trp Asp Gin Tyr CAC TGC TTT TGT His Cys Phe Cys

AAG

Lys 110 CAG TAT TTG TGG Gin Tyr Leu Trp,

CCG

Pro 115 ATA TTT CAT TAC Ile Phe His Tyr AAG GTT Lys Val 120 CCC GCT TCT Pro Ala Ser GCT TAT GTT Ala Tyr Val 140

GAC

Asp 125 GTC AAG AGT GTC Val Lys Ser Val AAT AGT CGG GAT Asn Ser Arg Asp TCA TGG, AAC Ser Trp Asn 135 ATG GAG GCA Met Glu Ala 435 CAC GTG AAC AAA His Val Asn Lys

GAG

Giu 145 TTT TCC CAG AAG Phe Ser Gin Lys

GTG

Val 150 *GTA ACC Val Thr 155 AAT CGT AGC A.AT Asn Arg Ser Asn GTA TGG ATA CAT GAC TAC CAT TTA ATG 531 Val Trp Ile His Asp Tyr His Leu Met 165

ACG

Thr 170 CTA CCG ACT Leu Pro Thr TTC TTG Phe Leu 175 AGG CGG GAT TTT Arg Arg Asp Phe TGT CGT Cys Arg 180 TTT AAA ATC Phe Lys Ile

GGT

Gly 185 TTT TTT CTG CAT AGC CCG TTT CCT TCC TCG GAG GTT Phe Phe Leu His Ser Pro Phe Pro Ser Ser Giu Val 190 195 TAC AAG ACC CTA Tyr Lys Thr Leu 200 CCA ATG AGA Pro Met Arg GAG CTC TTG AAG Giu Leu Leu Lys

GGT

Gly 210 CTG TTA AAT GCT GAT CTT ATC Leu Leu Asn Ala Asp Leu Ile 215 GGG TTC CAT Gly Phe His 220 CGA ATG TTT Arg Met Phe 235 ACA TAC GAT TAT Thr Tyr Asp Tyr

GC

Ala 225 CGT CAT TTT CTA Arg His Phe Leu TGT TGT AGT Cys Cys Ser 723 771 GGT TTG GAT GiyLeu Asp CAG TTG AAA AGG Gin Leu Lys Arg

GGG

Gly 245 TAG ATT TTC TTG Tyr Ile Phe Leu

GAA

Giu 250 TAT AAT GGA AGG Tyr Asn Gly Arg ATT GAG ATC AAG Ile Giu Ile Lys

ATA

Ile 260 AAG GCG AGC GGG Lys Ala Ser Gly

ATT

Ile 265 CAT GTT GGT CGA His Vai Gly Arg

ATG

Met 270 GAG TCG TAC TTG Giu Ser Tyr Leu CAG CCC GAT ACA Gin Pro Asp Thr AGA TTA Arg Leu 280 a

U

a a a a *9SaS~ a a.

a a CAA GTT CAA Gin Vai Gin 25 GTT GAT GAT Val Asp Asp 300 TTG GAG AAG 30 Leu Giu Lys 315

GAA

Giu 285 GTC CAA AAA CGT Val Gin Lys Arg AAG GAA ATC GTG Lys Giu Ile Vai CTA CTG GGA Leu Leu Giy 295 GTT TTA GCG Val Leu Ala TTG GAT ATA TTC Leu Asp Ilie Phe GGT GTG AAC TTC Giy Val Asn Phe

AAG

Lys 310 915 963 1011 TTA CTT AAA Leu Leu Lys CAC CCG AGT TGG His Pro Ser Trp

CAA

Gin 325 GGG CGT GTG GAA Giy Arg Vai Giu

AAG

Lys 35 330 GTG CAA ATC TTG Val Gin Ile Leu

AAT

Asn 335 CCT CTG CGC CGT Pro Leu Arg Arg

TGC

Cys 340 CAA GAG GTC GAT Gin Asp Vai Asp GAG 1059 Giu 345 ATC AAT GCC GAG Ile Asn Aia Glu

ATA

Ile 350 AGA ACA GTC TGT Arg Thr Val Cys

GAA

Giu 355 AGA ATC AAT AAC Arg Ile Asn Asn GAA CTG Giu Leu 360 1107 1155 GGA AGO CCG Gly Ser Pro

GGA

Giy 365 TAC GAG CCC GTT Tyr Gin Pro Vai

GTG

Val 370 TTA ATT GAT GGG Leu Ile Asp Gly CCC GTT TCG Pro Vai Ser 375 TTA AGT GAA AAA GOT GCT TAT Leu Ser Giu Lys Ala Ala Tyr 380 ACA CCG TTA GGT GAG GGA CTG Thr Pro Leu Arg Asp Gly Leu 395 400 TAT GCT ATG GCG GAT ATG GCA ATT GTT Tyr Ala Ile Ala Asp Met Ala Ile Vai 385 390 AAT CTT ATC CCG TAG GAG TAG GTC GTT Asn Leu Ile Pro Tyr Giu Tyr Val Vai 405 1203 1251

TOO

Ser 410 CGA CAA AGT GTT Arg Gin Ser Val

AAT

Asn 415 GAC CCA AAT COO AAT ACT Asp Pro Asn Pro Asn Thr CCA AAA. AAG AGC Pro Lys Lys Ser 1299 ATG CTA GTG GTC Met Leu Val Val

TCC

Ser 430 GAG TTC ATC GGT Glu Phe Ile Gly

GTT

Val 435 TCA CTA TOT TTA Ser Leu Ser Leu ACC GGG Thr Gly 440 1347 GCC ATA CGG Ala Ile Arg TAO GAC GCA Tyr Asp Ala I S 460 GTC AAC Val.-Asn 445 CCA TGG GAT Pro Trp Asp

GAG

Giu 450 TTG GAG ACA GCA Leu Giu Thr Ala GAA GCA TTA Giu Ala Leu 455 GCC CAC ATG Ala His Met 1395 1443 CTC ATG GOT COT Leu Met Ala Pro GAC CAT AAA GAA Asp His Lys Giu AAA CAG Lys Gin 475 TAT CAA TAO ATT Tyr Gin Tyr Ile TOO OAT GAT GTA Ser His Asp Val AAO TGG GOT AGO Asn Trp Ala Ser 1491 1539

TTO

Phe 490 TTT CAA GAT TTA Phe Gin Asp Leu CAA GOG TGO ATO Gin Ala Cys Ile

GAT

Asp 500 OAT TOT CGT AAA His Ser Arg Lys

OGA

Arg 505 geese.

5@ C C 5* 6* C. S

C

.5 0 *5e*

S

*SSS

C.

0 eeC.

25 TGC ATG.AAT TTA Cys Met Asn Leu

GGA

Gly 510 TTT GGG TTA GAT Phe Gly Leu Asp

ACT

Thr 515 AGA GTO GTC TTT Arg Val Val Phe TTG ATG Leu Met 520 AGA AGT TTA 30 Arg Ser Leu ATG GOT CAA Met Ala Gin 35 540

GOA

Ala 525 AGT TGG ATA AAG Ser Trp Ile Lys TOT TGG AAG AAT Ser Trp Lys Asn GOT TAT TCC Ala Tyr Ser 535 ACT GTT ACT Thr Val Thr 1587 1635 1683 AAT OGG 000 ATA Asn Arg Ala Ile TTG GAO TAT GAO Leu Asp Tyr Asp

GGC

Gly 550 COA TOT Pro Ser 555 ATO AGT AAA TOT Ile Ser Lys Ser OCA ACT Pro Thr 560 GAA GOT GTT Giu Ala Val

ATC

Ile 565 TCC ATG ATO AAO Ser Met Ile Asn 1731 1779

AAA

Lys 570 OTG TGC AAT GAT Leu Cys Asn Asp

OCA

Pro 575 AAG AAO ATG GTG Lys Asn Met Val ATO GTT AGT GGA Ile Val Ser Gly AGT AGA GAG AAA ATO TTG, GOA GTT GGT Ser Arg Giu Lys Ile Leu Ala Val Gly

TOG

Ser 595 GOG CGT GTG AGA Ala Arg Val Arg ACC OGO Thr Arg 600 1827 1875 CAT TGO ACT His Cys Thr

GAG

Giu 605 CAC GGA TAO TTT ATA AGO TGG GCG GOT GAT CAA GAA His Gly Tyr Phe Ile Arg Trp, Ala Gly Asp Gin Giu 610 615 TGG GA-A AG Trp Giu Thr 620 TGC GCA CGT GAG Cys Ala Arg Giu A-AT A-AT Asn Asn 625 GTC GGG TGG ATG GAT GGA A-AT 12 1923 Val Gly Trp Met 630 Asp Gly A-sn CTG AGG Leu Arg 635 GCG GTT ATG AAT Pro Val Met Asn TAT ACA GAA ACT Tyr Thr Giu Thr

ACT

Thr 645 GAG GGT TCG TAT Asp Gly Ser Tyr 1971

ATT

Ile 650 GA-A A-AG AAA GA-A Giu Lys Lys Glu GCA A-TG GTT TGG Ala Met Val Trp TAT GA-A GAT GCT Tyr Glu Asp Ala

GAT

Asp 665 2019 2067 AA-A GAT CTT GGG Lys Asp Leu Gly GAG GAG GCT A-AG Giu Gin Ala Lys

GAA

Giu 675 CTG TTG GAG CAT Leu Leu Asp His CTT GAA Leu Giu 680 A-AG GTG CTG Asn Vai Leu ATT GTA GA-A Ile Val Giu 700 A-AT GAG CCC GTT A-sn Giu Pro Val

GGA

Gly 690 GTG A-AT CGA ACA Vai Asn Arg Thr GGT GA-A TAG Gly Gin Tyr 695 GTT GTT ATG Leu Val Met GTT AAA CCA GAG Vai Lys Pro Gin CCC ATT AAT TAG Pro Ile Asn Tyr

GTT

Leu 710 2115 2163 2211 2259 ACA TTG Thr Phe 715 A-TA GGC ACT GAT Ile Gly Thr Asp AGA ATC TTT AAC Arg Ile Phe A-sn

TTA

Leu 725 A-AT TTC TTT AA-A A-sn Phe Phe Lys

TAT

Tyr 730 GAA TGC A-AT TAT Giu Cys A-sn Tyr

AGG

Arg 735 GGG TCA GTA A-A-A Gly Ser Leu Lys A-TA GTT GCA GAG Ile Vai A-ia Giu ATT TTT GCG TTC Ile Phe A-ia Phe GCT AAA A-AG GGA A-ia Lys Lys Giy

AA-A

Lys 755 GAG GCT GAT TTC Gin A-ia Asp Phe GTG TTG Val Leu 760 A-CG TTG A-AT Thr Leu A-sn GGA A-TA A-A-A Gly Ile Lys 780

GAT

Asp 765 A-GA AGT GAT GAA A-rg Ser Asp Giu

GA-C

Asp 770 ATG TTT GTG Met Phe Vai A-AG GGT GGG A-TA Lys Gly Arg Ile

ACT

Thr 785 A-AG A-AG A-AT TGA A-sn A-sn A-sn Ser GCC ATT GGG GAT A-ia Ile Giy Asp 775 GTG TTT A-CA TGG Val Phe Thr Cys 790 TTA A-AT GAT GTC Leu A-sn Asp Vai 2307 2355 2403 GTA GTG Val Val 795 GGA GAG AAA CCG Gly Giu Lys Pro

AGT

Ser 800 GCA GCT GAG TAG Ala A-ia Giu Tyr

TTT

Phe 805 2451

TCG

Ser 810 A-GA AGC TCC GGG A-rg Ser Ser Gly GTC AGC AAG GAA GGA Leu Ser A-sn Gin Gly T GATCCGGAAG 2495 CTTCTCGTGA TCTTTATGAG, TTA-AAAGTTT TCGAGTTTTT CTTGATCAAG ATTGATGGGA 2555 33 AAGTTGTTCA ATATGAACTT GTGTTCTTGG TTCTGGATTT TAGGGAGTCT ATGGATATAA

CATTTC

INFORM4ATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 820 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ile Leu Leu His Leu Met Pro Leu Gin Met Leu Pro Asn Arg Leu 2615 2621 0* 9 1 Ile Thr Thr Leu 65 Val1 35 Pro Leu Val Glu 145 Val Arg Val Val Met Giu 35 Cys Thr Giy Asp Ser Lys Thr Ser Tip Pro 115 Pro Asn 130 Phe Ser Tip Ile Asp Phe Ser Gly Ser Pro Thr Lys 100 Ile Se r Gin His Cys 180 Asn Gin Pro Phe Lys Met Leu Arg 70 Leu Leu Tip Asp Phe His Arg Asp Lys Vai 150 Asp Tyr 165 Arg Phe Leu Gly His 55 Ala Asp Gin Tyr Ser 135 Met His Lys Pro Ile 40 Tyr Asp Arg Tyr Lys 120 Tip Giu Leu Ile Ile 25 Ser Pro Val1 Phe Tyr 105 Val1 Asn Ala Met Gly 185 10 Ile Leu Gin Gly Asn 90 His Pro Ala Val Thr 170 Phe Ala Gly Pro Pro 75 Cys Cys Ala Tyr Th r 155 Leu Phe Arg Thr Leu Thr Val1 Phe Ser Val1 140 Asn Pro Leu Leu Arg Arg Giu Ala Cys Asp 125 His Arg Thr His Arg Val Phe Gin Val1 Lys 110 Val Val1 Ser Phe Ser 190 Leu Arg Ser Asp Phe Gin Lys Asn Asn Leu 175 Pro Thr Phe Ile Asp Val Tyr Ser Lys Tyr 160 Arg Phe Pro Ser Ser Glu Val Tyr Lys Thr Leu Pro Met Arg Asn Giu Leu Leu 195 200 205 Lys Gly Leu Leu Asn Ala Asp Leu Ile Gly Phe His Thr Tyr Asp Tyr 210 215 220 Ala Arg His Phe Leu Thr Cys Cys Ser Arg Met Phe Gly Leu Asp His 225 230 235 240 Gin Leu Lys Arg Gly Tyr Ile Phe Leu Glu Tyr Asn Gly Arg Ser lle 245 250 255 Glu Ile Lys Ile Lys Ala Ser Gly Ile His Val Gly Arg Met Glu Ser 260 265 270 Tyr Leu Ser Gin Pro Asp Thr Arg Leu Gin Val Gin Glu Val Gin Lys 275 280 285 Arg Ser Lys Glu Ile Val Leu Leu Gly Val Asp Asp Leu Asp Ile Phe 290 295 300 Lys Gly Val Asn Phe Lys Val Leu Ala Leu Glu Lys Leu Leu Lys Ser 305 310 315 320 His Pro Ser Trp Gin Gly Arg Val Glu Lys Val Gin Ile Leu Asn Pro 325 330 335 Leu Arg Arg Cys Gin Asp Val Asp Glu Ile Asn Ala Glu Ile Arg Thr 340 345 350 Val Cys Glu Arg Ile Asn Asn Glu Leu Gly Ser Pro Gly Tyr Gin Pro 30 355 360 365 Val Val Leu Ile Asp Gly Pro Val Ser Leu Ser Glu Lys Ala Ala Tyr 370 375 380 35 Tyr Ala Ile Ala Asp Met Ala Ile Val Thr Pro Leu Arg Asp Gly Leu 385 390 395 400 Asn Leu Ile Pro Tyr Glu Tyr Val Val Ser Arg Gin Ser Val Asn Asp 405 410 415 Pro Asn Pro Asn Thr Pro Lys Lys Ser Met Leu Val Val Ser Glu Phe 420 425 430 Ile Gly Val Ser Leu Ser Leu Thr Gly Ala Ile Arg Val Asn Pro Trp 435 440 445 Asp Glu Leu Glu Thr Ala Glu Ala Leu Tyr Asp Ala Leu Met Ala Pro 450 455 460 Asp Asp His Lys Glu Thr Ala His Met Lys Gin Tyr Gin Tyr Ile Ile 465 470 475 480 Ser His Asp Val Ala Asn Trp Ala Ser Phe Phe Gin Asp Leu Glu Gin 485 490 495 Ala Cys Ala yIle Asp His Ser Arg Lys Arg Cys Met Asn Leu Gly Phe Gly 500 505 510 9 Leu.

Lys Leu 545 Thr Asn Val Phe Asn 625 Tyr Met 35 Ala Val1 40 Ser 705 Arg Ser Lye Asep Met 530 Leu Glu Met Gly Ile 610 Aen Thr Val Lys Gly 690 Pro Ile Leu Gly Thr 515 Ser Asp Ala Val1 Ser 595 Arg Val1 Glu Trp Giu 675 Val Ile Phe Lys Lys 755 Arg Trp Tyr Val1 Phe 580 Ala Trp Gly Thr His 660 Leu Asn Asn Asn Gly 740 Gin Val Lys Asp Ile 565 Ile Arg Ala Trp Thr 645 Tyr Leu Arg Tyr Leu 725 Sle *Ala /a 1 Gly 550 Se r Val1 Val1 Gly met 630 Asp Giu Asp Thr Leu 710 Aen Val1 Asp Phe Ala 535 Th r Met Ser Arg Asp 615 Asp Gly Asp His Gly 695 Let; PhiE Alz Ph Leu I 520 Tyr Val IJ Ile Gly Thr 600 Gin Gly.

Ser Ala Leu 680 Gin Val Phe SGiu SVal 760 let er Lhr ksn krg 585 Arg Giu Asn Tyr Asp 665 Glu Tyr Met Lys Lys 745 Leu Arg S Met I Pro Lys 570 Ser His T rp Leu Ile 650 Lye Asn Ile Thr Tyr 730 Ile Thr er Ser Leu Arg Cys Giu Arg 635 Giu Asp Val Val Phe 715 Git PhE Lei Leu Gin 540 Ile Cys Giu Thr Thr 620 Pro Lys Leu Let Gi Ii( 1Cy~ SAl .i Asi Ala S 525 Aen A Ser L Aen P Lye Giu 605 *Cys Vail Lye *Gly Ala 685 Val Gly s Aen a Phe n Asp 765 er rg ~ys Lep Ele Us kla ,1et Glu Leu 670C Aer Ly, Thi Ty: Me' 75 Ar Trp I Ala I Ser P 5 Pro I 575 Leu Gly Arg Aen Thr 655 Giu 1Giu Pro Asp Arg 735 t Ala 0 g Ser le ie ro ~ys kia ryr Giu ILeu 640 Ala Gin Pro Gin Cys 720 Gly Lys Asp Giu Asp 770 Met Phe Val Ala Ile 775 Giy Asp Gly Ile Lye '780 Lys Gly Arg Ile 36 Thr Asn Asn Asn Ser Val Phe Thr Cys Val Val Gly Glu Lys Pro Ser 785 790 795 800 Ala Ala Glu Tyr Phe Leu Asn Asp Val Ser Arg Ser Ser Gly Cys Leu 805 810 815 Ser Asn Gin Gly 820 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAGCTTATGT TGCCATATAG AGTAG INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single o**o TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GTAGTTGCCA TGGTGCAAAT GTTC 24 37 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AGCTCTGCAG TGAGGTACCA INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid 25 STRANDEDNESS: single 25 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 35 GACGTCACTC CATGGTTCGA a INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs S: TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GTACCCTGCA GTGTGACCCT AGAC 24 38 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TCGATTCATA GAAGCTTAGA T 21 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 2207 base pairs TYPE: nucleic acid STRANDEDNESS: double 25 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Solanum tuberosum 35 STRAIN: Kardal (ix) FEATURE: NAME/KEY: CDS LOCATION: 161..1906 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 842..850 OTHER INFORMATION: /function= "putative glycosylationsite" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CTTTTCTGAG TAATAACATA GGCATTGATT TTTTTTCAAT TAATAACACC TGCAAACATT CCCATTGCCG GCATTCTCTG TTCTTACAAA AAAAAACATT TTTTTGTTCA CATAAATTAG 120 TTATGGCATC AGTATTGAAC CCTTTAACTT GTTATACAAT ATG GGT AAA GCT ATA Met Gly Lys Ala Ile 175 ATT TTT ATG ATT TTT ACT ATG TCT ATG AAT ATG ATT AAA GCT Ile Phe Met Ile Phe Thr Met Ser Met Asn Met Ile Lys Ala GAA ACT Glu Thr 223 TGC AAA TCC Cys Lys Ser

ATT

Ile GAT AAG GGT CCT Asp Lys Gly Pro ATC CCA ACA ACC Ile Pro Thr Thr CCT TTA GTG Pro Leu Val TAT GGC CAT Tyr Gly His ATT TTT CTT Ile Phe Leu GAA AAA GTT CAA Giu Lys Val Gin

GAA

Giu GCT GCT CTT CAA Ala Ala Leu Gin AAA GGG Lys Gly TTT GAT GCT AAA Phe Asp Ala Lys TTT GTT GAT ATG Phe Val Asp Met

TCA

Ser CTG AGA GAG AGT Leu Arg Giu Ser a.

CTT

Leu 70 TCA GAA ACA GTT Ser Glu Thr Val

GAA

Giu 75 GCT TTT AAT AAG Ala Phe Asn Lys CCA AGA GTT GTG Pro Arg Val Val

AAT

Asn GGT TCA ATA TCA Gly Ser Ile Ser AGT GAT TTG GAT Ser Asp Leu Asp TTT ATA GGT AGT Phe Ile Gly Ser TAC TTG Tyr Leu 100 AGT AGT CCT Ser Ser Pro GCT GAG CCT Ala Giu Pro 35 120 AAG GAT TTG GTT Lys Asp Leu Val

TAT

Tyr 110 GTT GAG CCT ATG Val Glu Pro Met GAT TTT GTG Asp Phe Val 115 GAG GTG AGG Giu Val Arg 511 GAA GGC TTT TTG Giu Gly Phe Leu

CCA

Pro 125 AAG GTG AAG, AAT Lys Val Lys Asn

TCT

Ser 130 GCA TGG Ala Trp 135 GCA TTG GAG GTG Ala Leu Giu Val TCA CTT TGG AAG AAT TTA AGT AGG AAA Ser Leu Trp Lys Asn Leu Ser Arg Lys 145 a a

GTG

Val1 150 GCT GAT CAT GTA Ala Asp His Val

TTG

ILeu 155 GAA AAA CCA GAG Glu Lys Pro Glu TAT ACT TTG Tyr Thr Leu CTT CCA Leu Pro 165 TTG AAA AAT CCA Leu Lys Asn Pro TAT TGG GAT TCT Tyr Trp, Asp Ser 185 ATT ATA CCG GGA TCG CGT TTT Ile Ile Pro Gly Ser Arg Phe 175 AAG GAG GTT TAT 703 Lys Glu Vai Tyr 180 TAT TGG GTA ATA AGG GGT TTG TTA GCA AGC AAA ATG 751 Tyr Trp Val Ile Arg Gly Leu Leu Ala Ser Lys Met 190 195 TAT GAA ACT Tyr Glu Thr 200 GCA AAA GGG ATT GTG ACT AAT CTG GTT TCT CTG ATA GAT Ala Lys Gly Ile Val Thr Asn Leu Val 205 Ser 210 Leu Ile Asp CAA TTT Gin Phe 215 GGT TAT GTT CTT Giy Tyr Val Leu

AAC

Asn 220 GGT GCA AGA GCA Gly Ala Arg Ala

TAC

Tyr 225 TAC AGT AAC AGA Tyr Ser Asn Arg

AGT

Ser 230 CAG CCT CCT GTC Gin Pro Pro. Val

CTG

Leu 235 GCC ACG ATG ATT Ala Thr Met Ile GAC ATA TTC AAT Asp Ile Phe Asn

CAG

Gin 245 895 ACA GGT GAT TTA Thr Gly Asp Leu TTG GTT AGA AGA Leu Vai Arg Arg

TCC

Ser 255 CTT CCT GOT TTG Leu Pro Ala Leu CTC AAG Leu Lys 260 GAG AAT CAT Giu Asn His GOT CAG GGA Ala Gin Gly 280 TGG AAT TCA GGA Trp Asn Ser Gly

ATA

Ile 270 CAT AAG GTG ACT His Lys Val Thr ATT CAA GAT Ile Gin Asp 275 ATG TGG AAT Met Trp, Asn 9* TCA AAC CAC AGC Ser Asn His Ser

TTG

Leu 285 AGT CGG TAC TAT Ser Arg Tyr Tyr

GOT

Ala 290 25 AAG CCC Lys Pro 295 CGT CCA GAA TCG Arg Pro Giu Ser

TCA

Ser 300 ACT ATA GAC AGT Thr Ile Asp Ser

GAA

Giu 305 ACA GCT TCC GTA Thr Ala Ser Val

CTC

30 Leu 310 CCA A.AT ATA TGT Pro Asn Ile Cys

GAA

Giu 315 AAA AGA GAA TTA Lys Arg Giu Leu CGT GAA CTG GCA Arg Giu Leu Ala

TCA

Se r 325 1039 1087 1135 1183 1231 1279 GCT GOT GAA AGT Ala Ala Giu Ser

GGA

Gly 330 TGG GAT TTC AGT Trp Asp Phe Ser

TCA

Se r 335 AGA TGG ATG AGC Arg Trp Met Ser AAC GGA Asn Gly 340 TCT GAT CTG Ser Asp Leu AAT GCA TTC Asn Ala Phe 360

ACA

Thr 345 ACA ACT AGT ACA Thr Thr Ser Thr

ACA

Thr 350 TCA ATT CTA CCA Ser Ile Leu Pro GTT GAT TTG Vai Asp Leu 355 CTA GOA AAT Leu Ala Asn CTT CTG AAG ATG Leu Leu Lys Met CTT GAC ATT GCC Leu Asp Ile Ala CTT GTT Leu Val 375 GGA GAA AGT AGC Gly Giu Ser Ser

ACG

Thr 380 GOT TCA CAT TTT Ala Ser His Phe

ACA

Thr 385 GAA GCT GCT CAA Giu Ala Ala Gin 1327

AAT

Asn 390 AGA CAG AAG GOT ATA AAC TGT ATC TTT TGG AAC GCA GAG Arg Gin Lys Ala Ile Asn Cys Ile Phe Trp Asn Ala Giu 395 400 ATG GGG Met Giy 405 1375 CAA TGG CTT GAT Gin Trp Leu Asp

TAC

Tyr 410 TGG CTT ACC AAC Trp Leu Thr Asn

AGC

Ser 415 GAC ACA TCT GAG Asp Thr Ser Giu GAT ATT Asp Ile 420 1423 TAT AAA TGG Tyr Lys Trp TTT GTT CCG Phe Vai Pro 440 GAA GAT TTG CAC CAG AAC AAG AAG TCA TTT GCC TCT AAT 1471 Asp Leu His Gin Asn 430 Lys Lys Ser Phe Aia Ser Asn 435 CTG TGG ACT GAA ATT TCT TGT TCA GAT Leuw.Trp Thr Giu Ile Ser Cys Ser Asp 445 AAT AAT ATC ACA 1519 Asn 450 Asn Ile Thr *ACT CAG Thr Gin 455 AAA GTA G3TT CAA Lys Vai Vai Gin CTC ATG AGC TCG Leu Met Ser Ser

GGC

Giy 465 TTG CTT CAG CCT Leu Leu Gin Pro 1567

GCA

Aia 470 GGG ATT GCA ATG Giy Ile Aia Met TTG TCT AAT ACT Leu Ser Asn Thr

GGA

Giy 480 CAG CAA TGG GAT Gin Gin Trp Asp 1615

S

S

*9 9 9 CCG AAT GGT TGG Pro Asn Giy Trp

CCC

Pro 490 CCC CTT CAA CAC Pro Leu Gin His

ATA

Ile 495 ATC ATT GAA GGT Ile Ile Giu Giy CTC TTA Leu Leu 500 25 AGG TCT GGA Arg Ser Giy CGC TGG TTA 30 Arg Trp, Leu 520 TAT GAA AAA Tyr Giu Lys 535 GAA TAT ATG Giu Tyr Met 550

CTA

Leu 505 GAA GAG GCA AGA Giu Giu Aia Arg

ACC

Thr 510 TTA GCA AAA GAG Leu Aia Lys Asp ATT GCT ATT Ile Aia Ile 515 GGT GCT ATG Gly Aia Met AGA ACT AAC TAT Arg Thr Asn Tyr

GTG

Val1 525 ACT TAG AAG, AAA Thr Tyr Lys Lys

AC

Th r 530 1663 1711 1759 1807 1855 1903 TAT GAT GTG Tyr Asp Val TGG CAA AG Ser Gin Thr 555

AGA

Thr 540 AAA TGT GGA GGA Lys Cys Giy Aia

TAT

Tyr 545 GOA GOT GOT GOT Gly Giy Giy Giy GOT TTC GGA TGO Gly Phe Giy Trp

TCA

Ser 560 AAT GGC GTT GTA Asn Giy Vai Val

CTG

Leu 565 GCA CTT GTA GAG Ala Leu Leu Giu

GAA

Giu 570 TTT GGA TGG CCT Phe Gly Trp, Pro

GAA

Giu 575 GAT TTG AAG ATT Asp Leu Lys Ile GAT TGC Asp Cys 580 TAATGAGCAA GTAGAAAAGC CAAATGAAAC ATGATTGAGT TTTATTTTGT TGTTTTGTTA AAATAAGGTG CAATGGTTTG CTGATAGTTT ATGTTTTGTA TTACTATTTC ATAAGGTTTT TGTACGATAT CAAGTGATAT TAGGATGAAG TATGTCGTTG GGACTCTTCA AATGGOATTT TGGAAAAATA ATGCAGTTTT GGAGAATGGG ATAACATAGA GGATGTATGG ATGTAAATTG TAAAGAGCTT ACTATATTAA GTAAAAGAAA GATGATTGGT GTGGTTTAA.A AAAA.AAAAAA 1963 2023 2083 2143 2203 AAAA 2207 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS:.

LENGTH: 581 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Giy Lys Ala Ile Ile Phe Met Ile Phe Thr Met Ser Met Asn Met 1

S.

1 Ile Thr Gin Ser Pro Ile Pro Asn 40 Asn 145 Tyr Phe Lys Thr Thr 50 Leu Arg Giy Met Ser 130 Leu Thr Lys Ala Pro Tyr Arg Val1 Ser Asp 115 Giu Ser Leu Glu Glu Leo Giy Glu Val1 Tyr 100 Phe Val Arg Leu Val1 180 5 Thr Val1 His Ser Asn Leu Val1 Arg Lys Pro 165 Tyr Cys Ile Lys Leu 70 Giy Ser Ala Ala Val1 150 Leu Tyr Lys Phe Gly 55 Se r Se r Ser Giu Trp 135 Ala Lys Trp, Ser Leu 40 Phe Giu Ile Pro Pro 120 Ala Asp Asn Asp Ile Asp 25 Giu Lys Asp Ala Thr Val Ser Lys 90 Asp Lys 105 Glu Gly Leu Glu His Val Pro Val 170 Ser Tyr 185 Lys Val1 Lys Giu 75 Ser Asp Phe Vai Leu 155 Ilie Gly Gin Leu Ala Asp Leu Leu His 140 Giu Ile P ro Glu Phe Phe Leu Val Pro 125 Se r Lys Pro Val Ala Val1 Asn Asp Tyr 110 Lys Leu Pro Giy Ile Ala Asp Lys Giy Val1 Val1 T rp Glu Sei 171 Pro Leu Met Leo Phe Giu Lys Lys Leu 160 Arg Trp Val Ile Arg Gly Leu 190 Thr Asn Leo Leo Ala Ser 195 Lys Met Tyr Giu Thr 200 Ala Lys Gly Ile Val1 205 43 Val Ser Leu Ile Asp Gin Phe Giy Tyr Vai 210 215 Leu Asn Gly Ala Arg Ala 220 0 S Tyr 225 Asp Pro.

Vai Tyr Giu 305 Arg 25 Trp Leu 30 Ala Thr 35 385 Asn 40 Thr Ser Asp Giy 465 ryr Ile Ala Thr Ala 290 Thr Giu Met Pro Phe 370 Giu Ala Ser Phe Asn 450 Leu Ser Phe Leu Ile 275 Met Ala Leu Ser Val1 355 Leu Ala Giu Giu Ala 435 Asn Leu Asn Asn Leu.

260 Gin Trp Ser Ala Asn 340 Asp Ala Ala Met Asp 420 Ser Ile Gin Arg Gin 245 Lys Asp Asn Val1 Ser 325 Gly Leu Asn Gin Gly 405 Ile Asn Thr Pro Ser 230 Thr Glu Aia Lys Leu 310 Ala Ser Asn Leu Asn 390 Gin Tyr Phe Thr Ala 470 Gin Gly Asn Gin Pro 295 Pro Ala Asp Aia Val1 375 Arg Trp Lys Val1 Gin 455 Gly ?ro lksp Gly 280 Arg Asn Giu Leu Phe 360 Giy Gin L.eu Trp Pro 440 Lys Ile Pro Leu Phe 265 Ser Pro Ile Ser Thr 345 Leu Giu Lys Asp Giu 425 Leu Val Ala Val Asn 250 Trp Asn Giu Cys Gly 330 Thr Leu Ser Ala Tyr 410 Asp Trj Va) Met Leu 2 235 Leu N Asn His Ser Giu 315 Trp Th r Lys Ser Ile 395 Trp, Leu Thr Gin Thr lia al1 3er Ser Ser 300 Lays Asp Se r met Thr 380 Asn Leu His Gl.

Se 46( Le~ Thr b Arg Gly Leu 285 Thr Arg Phe Thr Giu 365 Ala Cys Thr Gin 445 Leu .i Ser det I k.rg E Ile 270 Ser Ile Glu Ser Th r 350 Leu Ser Ile Asn Asn 430 Ser Met Asn lie ~er fis krg k.sp Leu Ser 335 Ser Asp His Phe Ser 415 Lys Cys Sex Thi Val1 240 Leu Lys Ty~r Ser Tyr 320 Arg Ile Ile Phe Trp 400 Asp Lys Ser Ser Gly 475 480 Gin Gin Trp Asp Phe Pro Asn Gly Trp, Pro Pro Leu Gin His Ile Ile 485 490 495 Ile Glu Gly Lys Asp Ile 515 Lys Thr Gly Leu Leu 500 Ala Ile Ala Met Arg Ser Gly Arg Trp Leu 520 Tyr Glu Lys 535 Glu Tyr Met Leu 505 Arg Tyr Ser Glu Glu Ala Arg Thr Leu Ala 510 Thr Asn Tyr Val Thr Tyr Lys 525 Asp Val Thr Lys Cys Gly Ala 540 Gin Thr Gly Phe Gly Trp Ser 530 Tyr 545 Asn Gly Gly Gly Gly 555 Glu Glu Phe 560 Gly Trp Pro Glu Asp 575 Gly Val Val Leu 565 Leu Leu 570 Leu Lys Ile Asp Cys 580 *9 V99.

C.0

C

C

C

C

C

INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 30 (iii) HYPOTHETICAL: YES (ix) FEATURE: NAME/KEY: modified_base 35 LOCATION: 6 OTHER INFORMATION: /mod_base= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: OTHER INFORMATION: /mod_base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGYGGNMGMT TYRWNGARKT MTAYKRYTGG GAC INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 3 OTHER INFORMATION: /modbase= i (ix) FEATURE: NAME/KEY: modified base LOCATION: 6 OTHER INFORMATION: /mod base= i (ix) FEATURE: NAME/KEY: modified base LOCATION: 9 OTHER INFORMATION: /mod base= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 12 OTHER INFORMATION: /mod base= i *s (ix) FEATURE: NAME/KEY: modified base LOCATION: OTHER INFORMATION: /mod base= i (ix) FEATURE: NAME/KEY: modified base LOCATION: 21 I* 35 OTHER INFORMATION: /mod base= i ee (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: o 40 GTNCCNGGNG GNCGNTTYRW NGARKT 26

S

INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES

I

46 (ix) FEATURE: NAME/KEY: modified base LOCATION: 3 OTHER INFORMATION: /mod_base= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 9 OTHER INFORMATION: /modbase= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 12 OTHER INFORMATION: /mod base= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: OTHER INFORMATION: /modbase= i (ix) FEATURE: NAME/KEY: modified base LOCATION: 18 OTHER INFORMATION: /mod base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GGNGGYTGNS WNCGNYRNAG RTARTA 26 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 4 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) FEATURE: NAME/KEY: modified base LOCATION: 1 OTHER INFORMATION: /mod base= i (ix) FEATURE: NAME/KEY: modified base LOCATION: 7 OTHER INFORMATION: /mod base= i 47 (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 19 OTHER INFORMATION: /modbase= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 22 OTHER INFORMATION: /mod base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: NSCRTTNRYC CATCCRAANC CNTC 24 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO 30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CGAAACGGGC CCATCAATTA INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single 40 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TCGATGAGAT CAATGCCGAG 48 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CCATCCTAAT ACGACTCACT ATAGGGC 27 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single 25 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 35 CACAACAGGC TGGTATCCCG INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: 40 LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CAATAACGAA CTGGGAAGCC 2 49 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ACTCACTATA GGGCTCGAGC GGC 23 INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single 25 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: modified_base LOCATION: 4 35 OTHER INFORMATION: /mod_base= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 6 OTHER INFORMATION: /mod base= i (ix) FEATURE: NAME/KEY: modified base LOCATION: 9 OTHER INFORMATION: /mod base= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: OTHER INFORMATION: /modbase= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GAYNTNATNT GGRTNCAYGA YTAYCA 2 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: modified base LOCATION: 3 OTHER INFORMATION: /mod_base= i (ix) FEATURE: NAME/KEY: modified base LOCATION: 6 OTHER INFORMATION: /modbase= i 25 (ix) FEATURE: NAME/KEY: modified_base LOCATION: 12 OTHER INFORMATION: /mod_base= i 30 (ix) FEATURE: NAME/KEY: modified_base LOCATION: 18 OTHER INFORMATION: /mod_base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: •CCNACNGTRC ANGCRAANAC INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO 51 (ix) FEATURE: NAME/KEY: modified base LOCATION: 2 OTHER INFORMATION: /modbase= i (ix) FEATURE: NAME/KEY: modified base LOCATION: OTHER INFORMATION: /modbase= i (ix) FEATURE: NAME/KEY: modified base LOCATION: 8 OTHER INFORMATION: /mod base= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 14 OTHER INFORMATION: /mod_base= i (ix) FEATURE: S* NAME/KEY: modified base LOCATION: OTHER INFORMATION: /modbase= i **25 (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 23 OTHER INFORMATION: /modbase= i *g (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 35 TNGGNTKNTT YYTNCAYAYN CCNTTYCC 28 INFORMATION FOR SEQ ID NO: 24: S" SEQUENCE CHARACTERISTICS: 40 LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: modified base LOCATION: 6 OTHER INFORMATION: /modbase= i (ix) FEATURE: NAME/KEY: modified_base LOCATION: 9 OTHER INFORMATION: /modbase= i (ix) FEATURE: NAME/KEY: modified_base LOCATION: 18 OTHER INFORMATION: /mod_base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: TGRTCNARNA RYTCYTTNGC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: modified_base LOCATION: 9 OTHER INFORMATION: /mod_base= i (ix) FEATURE: 35 NAME/KEY: modified_base LOCATION:. 12 OTHER INFORMATION: /mod_base= i (ix) FEATURE: 40 NAME/KEY: modifiedbase LOCATION: OTHER INFORMATION: /mod_base= i (ix) FEATURE: NAME/KEY: modified_base LOCATION: 18 OTHER INFORMATION: /mod_base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCRTGYTCNG CNSWNARNCC 'F 53 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 6 OTHER INFORMATION: /mod_base= i (ix) FEATURE: NAME/KEY: modifiedbase LOCATION: 17 OTHER INFORMATION: /mod_base= i 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: TCRTCNGTRA

ARTCRTCNCC

INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single 35 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (ix) FEATURE: NAME/KEY: modified_base LOCATION: 3 OTHER INFORMATION: /mod_base= i (ix) FEATURE: NAME/KEY: modified_base LOCATION: 6 OTHER INFORMATION: /mod_base= i (ix) FEATURE: NAME/KEY: modified_base LOCATION: 1 54 OTHER INFORMATION: /mod-base= i (ix) FEATURE: NAME/KEY: modified-base LOCATION: 21 OTHER INFORMATION: /mod-base= i (xi) SEQUENC E DESCRIPTION: SEQ ID NO: 27: GYNACNARRT TCATNCCRTC NC 22

Claims (14)

1. A chimaeric gene encoding a bipartite trehalose synthesizing enzyme.

2. A chimaeric gene according to claim 1, said gene being plant-derived and plant-expressible.

3. A chimaeric gene according to claim 1 or 2, said gene comprising, in sequence: a transcription initiation region obtainable from a gene, preferentially I 1 expressed in a plant part, particularly the patatin gene from Solanum tuberosum; 15 (ii) a 5'-untranslated leader; (iii) an open reading frame encoding a bipartite trehalose synthesizing enzyme; and (iv) downstream of said open reading frame, a transcriptional terminator region, wherein said transcriptional terminator region is preferably obtainable from the proteinase inhibitor-II gene of Solanum tuberosum.

4. A vector comprising a chimaeric plant expressible gene according to any of claims 1 to 3. *9 9

5. A recombinant plant genome comprising a chimaeric gene according to any of claims 1 to 3.

6. A plant cell having a recombinant genome according to claim

7. A plant or a part thereof, consisting essentially of cells according to claim 6, preferably a plant from the species Solanum tuberosum.

8. A plant part according to claim 7, which is a tuber or a micro-tuber. W:anice dpm\10085dalms.doc

9. A process for obtaining trehalose, comprising the steps of: growing a plant cell according to claim 6, or cultivating a plant according to claim 7, or cultivating a plant part according to claim 7 or 8; and (ii) extracting trehalose from said plant cells, plants or parts.

A process for producing trehalose in plant cells, said plant cells having been genetically altered so as to contain a gene coding for a bipartite trehalose synthesizing enzyme in a plant expressible form and said plant cells being capable of producing trehalase, wherein said process comprises growing plant cells having genetic information required for the production of trehalose and trehalase, or cultivating a plant or a part thereof comprising such plant cells, characterized in that said plant cells are grown, or said plant or a part thereof is cultivated in the presence of a trehalase inhibitor. *e .9 15

11. A process for obtaining trehalose, comprising the steps of: producing trehalose in plant cells, a plant or a part thereof, according to claim 10; and (ii) separating or extracting trehalose from said plant cells, plant or part thereof.

12. Trehalose when produced by a process according to any one of claims 9, or 11. 9

13. A chimaeric gene according to claim 1 substantially as hereinbefore described with reference to any one of the examples.

14. A process according to any one of claims 9, 10 or 11 substantially as hereinbefore described with reference to any one of the examples. DATED: 28 July, 2000 PHILLIPS ORMONDE FITZPATRICK Attorneys for: MOGEN INTERNATIONAL N.V. W:1janicepdpm\1 085daims.doc