DNA sequences encoding carbohydrate polymer synthesizing enzymes and method for producing transgenic plants

BACKGROUND OF THE INVENTION
This invention relates to nucleotide sequences encoding fructan synthesizing enzymes, a recombinant DNA sequence comprising one or more of said nucleotide sequences, a method for producing a genetically transformed host organism showing a modified fructan profile, and transformed plants or plant parts showing said modified fructan profile.
Fructans refer to a group of carbohydrate compounds in which one or more fructosyl-fructose linkages constitute a majority of the linkages. Fructans are fructosepolymers with usually, but not necessarily, one terminal glucosylunit [G-(F).sub.n, G=optional, n.gtoreq.2]. The fructosyl-fructose linkages in fructans are of the .beta.-2,6 or .beta.-2,1 linkage type. Fructans with predominantly .beta.-2,6 fructosyl-fructose linkages are usually called levan(s). Fructans with predominantly .beta.-2,1 fructosyl-fructose linkages are usually called inulin(s).
Fructan biosynthesis is common in several bacterial, fungal and algal families and also in specific plant families, such as the Liliaceae (e.g. Allium cepa), Poaceae (e.g. Lolium perenne) and Asteraceae (e.g. Helianthus tuberosus). The function of fructans in bacteria and fungi is poorly understood. It has been suggested that fructans act as extracellular stores of carbohydrate that can be mobilized during periods of carbohydrate stress (Jacques, 1993). In plants, fructans can function as reserve carbohydrates which serve as a source of carbon for (re)growth (Meier and Reid, 1982). In fructan-storing crops, tructan synthesis is restricted not only to specific organs (e.g. the stems or tubers of H. tuberosus, the bulbs of Allium spp, the leaf bases and stems of grasses) but also specific cell types within these organs (usually the parenchyma cells). In these specific cell types, the vacuole is probably the location of both fructan biosynthesis and storage (Darwen and John, 1989; Wagner et al., 1983).
In bacteria, examples of fructan synthesizing bacteria are Streptococcus mutans and Bacillus subtilus, the biosynthesis of fructans from sucrose is catalysed by only one enzyme: levansucrase (EC 2.4.1.10) in B. subtilus (Dedonder 1966) and levansucrase, but also called fructosyltransferase, (FTF, EC 2.4.1.10) in S. mutans (Carlsson, 1970). Bacterial fructan synthesis proceeds via the direct transfer of fructose from a donor-sucrose (G-F) to sucrose or other acceptor molecules according to the following reversible reaction:
n(G-F)+acceptor.fwdarw.n(G)+acceptor-(F).sub.u, (1)
where n may be larger than 10.000
Water, hexoses, sucrose, oligosaccharides and levan may act as acceptor molecules for fructosyl units from sucrose (fructosyl donor).
Bacterial DNA sequences encoding FTF in S. mutans and levansucrase in B. subtilus are already described in the literature (Sato and Kuramitsu, 1986; Steinmetz et al. 1985). Bacterial genes from several sources were used to transform specific host plants which normally cannot synthesize fructans, thereby inducing fructan synthesis (see for example: Van der Meer et al., 1994; Ebskamp et al., 1994). A method to enhance the solid content of tomato fruits, using the levansucrase gene from B. subtilus and the dextransucrase gene from Leuconostoc mesenretoides is described in application WO 89/12386. A method to modify the fructan pattern in plants which normally cannot synthesize fructans, using the levansucrase-encoding ftf gene from S. mutans and the levansucrase-encoding SacB gene from B. subtilus is described in applications NL A 9300646 and WO 94/14970. The use of a levansucrase-encoding DNA sequence from Erwinia amylovora, which after integration in the host plant genome leads to the synthesis of levans, is described in DE 4227061 A1 and WO A 9404692. In all said applications, transgenic plants are described which are transformed with levansucrase genes from bacteria. Accordingly, these transgenic plants synthesize and accumulate fructans structurally comparable to those synthesized by the donor bacteria (Van der Meer et al., 1994; Ebskamp et al., 1994).
The present application differs from said applications in that it is related to fructosyltransferase-encoding DNA sequences derived from plants. These enzymes are structurally different from bacterial enzymes since there is no significant homology at the amino acid level and DNA level. Besides, the mechanism of fructan biosynthesis in plants is essentially different from that in bacteria. In contrast to fructan biosynthesis in bacteria, the formation of fructans in plants is mediated by more than one enzyme. For example, in Relianthus tuberosus (the Jerusalem Artichoke), fructan biosynthesis is catalysed by two enzymes: sucrose:sucrose fructosyltransferase (SST, EC 2.4.1.99) and fructan:fructan fructosyltransferase (FFT, EC 2.4.1.100). The SST and FFT from H. tuberosus are involved in the synthesis of .beta.-2,1 linked fructans (inulin) and are therefore also designated as 1-SST and 1-FFT. 1-FFT has been purified from tubers of H. tuberosus (Luscher et al., 1993, Koops and Jonker, 1994). The purification of SST has proven more difficult to achieve. A putative SST has been purified, at a very low yield, from several plant sources (Shiomi and Izawa, 1980; Praznik et al., 1990; Angenent et al., 1993). However, in none of these studies the purity of the enzyme has convincingly been shown. Furthermore, it has not conclusively been shown in these studies that the isolated enzyme does not represent an invertase.
Large quantities of 1-SST and 1-FFT have now been purified up to homogeneity from tubers of H. tuberogus (1-FFT: Koops and Jonker 1994) and their reaction mechanisms extensively investigated. 1-SST from H. tuberosus catalyses the initial step of fructan biosynthesis, the synthesis of the trisaccharide 1-kestose (1-[G-(F).sub.2 ]) from two molecules of sucrose (G-F), according to the following reaction:
G-F+G-F.fwdarw.1-[G-(F).sub.2 ]+G, (2)
wherein G-F=sucrose, -F=fructosylunit, -G=glucosylunit, G=glucose
1-SST can also catalyse the formation of the tetrasaccharide 1,1-[G-(F).sub.3 ] and pentasaccharide 1,1,1-[G-(F).sub.4 ] (FIG. 3A). Therefore, 1-SST activity can be described by the following general reaction:
G-(F).sub.n +G-(F).sub.m .fwdarw.G-(F).sub.n-1 +G-(F).sub.m+1, 1.ltoreq.n.ltoreq.3, 1.ltoreq.m.ltoreq.3 (3)
It has also been found that 1-SST from H. tuberosus to some extent can catalyse the transfer from a fructosyl unit from G-(F).sub.n, 1.ltoreq.n.ltoreq.3, onto water.
The second enzyme, 1-FFT, catalyses the formation of fructans with a higher degree of polymerization. This enzyme catalyses a polymerization reaction by the transfer of fructosyl units between trisaccharides, tetrasaccharides and larger fructose polymers according to the following general reaction:
G-(F).sub.n +G-(F).sub.m .fwdarw.G-(F).sub.n-1 +G-(F).sub.m+1, n.gtoreq.2, m.gtoreq.2 (4)
It has also been found that 1-FFT catalyses the transfer of fructosyl units between sucrose (G-F) and galactose (Gal)-containing carbohydrates [(Gal).sub.n -G-F], also called galactans. For example, 1-FFT can catalyse the transfer of a fructosyl unit from G-(F).sub.2 onto raffinose (Gal-G-F) which results in the formation of [Gal-G-(F).sub.2 ]. It cannot be excluded that both 1-SST and 1-FFT from H. tuberosus can use other substrates as fructosyl acceptor.
Although 1-SST and 1-FFT have some overlapping activity--both enzymes can catalyse the formation of tetra and pentasaccharides (reactions 3 or 4)--1-SST and 1-FFT are distinctly different enzymes. The 1-SST and 1-FFT proteins have different physical properties and are encoded by different genes. 1-SST and 1-FFT have essentially different enzymic properties. 1-FFT is not able to catalyse the initial step of fructan synthesis (reaction 2), whereas 1-SST is not able to catalyse the formation of fructan polymers with a degree of polymerization higher then 5 [G-(F).sub.n, n>4]. In conclusion, with 1-SST activity alone, it is only possible to synthesize oligofructans from sucrose with a degree of polymerization of up to 5 [G-(F).sub.n, 2.ltoreq.n.ltoreq.4]. To synthesize fructans with a higher degree of polymerization and using sucrose as a substrate, both 1-SST and 1-FFT are needed. With 1-FFT activity alone, it is not possible to synthesize fructans from sucrose. It was found by the present inventors that protein fractions containing purified 1-SST as well as purified 1-FFT could use sucrose as a sole substrate for the synthesis of fructans with a degree of polymerization of at least 15 [G-(F).sub.14, FIG. 3B].
Bacterial fructans differ from fructans in plants with respect to the degree of polymerization and branching type and, consequently, in chemical and physical properties. In general, fructans from plants are assembled from less than 1000 fructosylunits. Fructans from H. tuberosus are assembled from less than 100 fruccosylunits. Fructans synthesized by bacteria may comprise more than 10.000 fructosyl units. Plant and bacterial fructans therefore differ in their possible applications. For fructans with a relatively low degree of polymerization, such as those isolated from Asteraceae (e.g. Jerusalem Artichoke, chicory or dahlia), an application as phosphate substitute in calcium binding agents and detergents has already been worked out (WO91/17189). Other applications are related to the organoleptical properties of fructans. The sweetening strength of fructans G-(F).sub.n decreases with an increasing degree of polymerization (increasing n-value). The sweetening strength of the oligofructans G-(F).sub.2 and G-(F).sub.3 approximates that of sucrose (G-F). The very long chain fructans such as those occurring in bacteria are not sweet at all. Very short chain fructans, such as those synthesized by sucrose:sucrose fructosyltransferase can therefore be used as sweeteners with the additional advantage that these sweet-tasting fructans are non-cariogenic and can withstand digestion in the digestive tract of humans, which opens possibilities for use as a low caloric sweetener. The short chain fructans, and also the longer chain fructans, can be used as the hydrophilic moiety of biosurfactants.
In contrast to the bacterial genes encoding levansucrase, which have already been cloned, the genes encoding SST and FFT have not been isolated before from plants. We found that the SST and FFT-encoding genes from plants, at the amino acid level, have no significant similarity to the known levansucrases and, at the DNA level, have no significant degree of homology to the levansucrase genes. For this reason it has not been possible to isolate the fructosyl transferase genes from plants using heterologous levansucrase probes from bacteria. It has also not been possible to isolate SST and FFT-encoding genes from plants using the amino acid sequences of the purified SST and FFT enzymes and their deduced oligonucleotide primers. The reason for this is that, although methods have been described for the purification of fructosyl transferases from plants, it has not been possible so far to obtain SST and FFT enzymes in sufficiently large amounts and with sufficiently high degrees of purity.
SUMMARY OF THE INVENTION
The object of this invention is to provide nucleotide sequences encoding SST and FFT.
Another object of this invention is to provide nucleotide sequences, obtained by recombination or mutagenesis of nucleotide sequences encoding SST or FFT, which encode enzymes having fructosyltransferase activity.
Yet another object of this invention is to provide a method for transforming non-fructan synthesizing crops into fructan-synthesizing crops by introduction of the SST and/or FFT-encoding genes.
Yet another object of this invention is to (partly) switch off the fructan synthesis in crops which normally synthesize fructans.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Analysis of Mono Q fractions of 1-SST purification and Superdex HR 75 fractions of 1-FFT purification on SDS-PAGE. Lane 1, Molecular Weight marker (MW is given in kDa); lane 2, Mono Q fraction with 1-SST activity; lane 3, Superdex HR 75 fractions with 1--FFT activity.
FIGS. 2A-2C. RP-HPLC separations of tryptic digests of: a. the 25 kDa polypeptide of 1-SST (A); b. the 55 kDa polypeptide of 1-SST (B); c. the 70 kDa 1-FFT polypeptide (C). Free eluting peptide fractions indicated with arrows were collected manually and subjected to amino acid sequencing.
FIGS. 3A-3B. HPAEC separations of oligofructans synthesized from sucrose by purified 1-SST (A) and by a mixture of purified 1-SST and purified 1-FFT (B). The reactions were performed in 100 mol m.sup.-3 sucrose, 2 mol m.sup.-3 DTT, 10 mol m.sup.-3 citrate/phosphate buffer, pH 5.0, o.o1% Na-azide at 25.degree. C. Reaction time was 80 h. Reaction was stopped by boiling the reaction mixture for 5 min. Rhamnose was used as an internal standard.
FIGS. 4A-4B. Nucleotide sequence and deduced amino acid sequence of the isolated 1-SST (A) and 1-FFT (B) cDNA. The amino acids determined by amino acid sequencing (see also Table 1) of the purified 1-SST and 1-FFT proteins are underlined.
FIGS. 5A-5B. Gene constructs pVS1 (A) and pVF1 (B). The chimeric constructs consist of the enhanced CaMV35S promoter with ALMV translational enhancer (X), the coding sequence of sst (Y) or fft (W), and the nos termination signal (Z). Restriction sites which were used in the cloning procedure are indicated.
FIG. 6. TLC analysis of fructans in transgenic potato plants harboring the pSF1 construct. TLC plates were developed twice in 90% aqueous acetone. S=sucrose standard, G=glucose standard, F=fructose standard, H=standard fructan mixture from tubers of H. tuberosus, N=Neosugar standard, K=G-(F).sub.2 -standard. No. 1-6 represent individual potato plants harboring the pVS1 construct. C is control plant harboring the AGLO construct.
FIG. 7. HPAEC separations of carbohydrates extracted from leaves of Petunia hybrida harboring the pVS1 construct (a), a standard fructan mixture extracted from H. tuberosus tubers (b), and carbohydrates from leaves of the control Petunia harboring the AGL0 construct (c).
FIG. 8. HPAEC separations of carbohydrates extracted from leaves of potato (Solanum tuberosum) harboring the pVS1 construct (a), a standard fructan mixture extracted from H. tuberosus tubers (b), and carbohydrates from leaves of the control potato harboring the AGL0 construct (c).
FIG. 9. TLC analysis of fructans synthesized from G-(F).sub.4 by protein extracts from leaves of transgenic potato plants harboring the pVF1 construct. TLC plates were developed twice in butan-1-ol, propan-2-ol and water (3:12:4, v/v). S=sucrose standard; N=Neosugar standard, H=standard fructan mixture from tubers of H. tuberosus. No. 1-8 represent different individual transgenic potato plants harboring the pVF1 construct. C.sub.1 and C.sub.2 are control plants harboring the AGL0 construct.

DETAILED DESCRIPTION OF THE INVENTION
Accordingly, this invention provides a DNA fragment having a nucleotide sequence SEQ ID. No. 1, as shown in FIG. 4A, or a homologous sequence having a degree of homology of at least 70% which encodes 1-sucrose:sucrose fructosyltransferase. Further, this invention provides a DNA fragment having a nucleotide sequence SEQ ID. No. 3 as shown in FIG. 4B or a homologous sequence having a degree of homology of at least 70% which encodes 1-fructan:fructan fructosyltransferase.
1-Sucrose:sucrose fructosyltransferase (1-SST) and 1-fructan:fructan fructosyltransferase (1-FFT) were purified from tubers of Helianthus tuberosus. Purified enzymes were cleaved into peptides by tryptic digestion, and the resulting peptide mixtures were separated by HPLC. N-terminal amino acid sequencing was performed for selected peptides. Amino acid sequences specific for 1-SST and 1-FFT were used to design degenerated oligonucleotide primers specific for 1-SST and 1-FFT, respectively, for use in RT-PCR. PCR was performed using cDNA as template, a tail-specific primer and the degenerated primers specific for either 1-SST or 1-FFT. First-strand cDNA was synthesized from poly(A).sup.+ RNA isolated from tubers from H. tuberosus. With the 1-SST-specific primer, RT-PCR resulted in a 450 bp specific fragment. With the 1-FFT-specific primer, RT-PCR resulted in a 800 bp specific fragment. The 450 and 800 bp PCR fragments were subsequently used to screen a cDNA library made from H. tuberosus tubers to isolate the full length cDNA sequences encoding 1-SST and 1-FFT, respectively.
The SST and FFT-encoding sequences from plants of the present invention induce, after insertion into the genome of a host organism, for example a plant, chances in the concentrations of carbohydrates containing at least one fructosyl unit (sucrose, oligofructans, fructans or galactans), or cause a change in the degree of polymerization of oligofructans, fructans or galactans. The present invention is related to said carbohydrates, since sucrose is the substrate for SST, oligofructans are products of SST, oligofructans and fructans with a higher degree of polymerization are substrates and products of FFT. Besides, said fructosyltransferase enzymes can also perform transfructosylation reactions with galactans of the raffinose series as fructosylacceptor.
The present invention includes DNA sequences which are at least 70% identical to the 1-SST-encoding sequence from H. tuberosus, irrespective of whether the homologous sequences are derived from other plant sources, or obtained by mutagenesis of fructosyltransferase-encoding sequences from plant sources or from microorganisms. It is preferred that the degree of homology is at least 80%, more preferred that the degree of homology is at least 85% and still more preferred that the degree of homology is at least 90%. It is most particularly preferred that the degree of homology is at least 95%.
The present invention includes DNA sequences which are at least 70% identical to the 1-FFT-encoding sequence from H. tuberosus, irrespective of whether the homologous sequences are derived from other plant sources, or obtained by mutagenesis of fructosyltransferase-encoding sequences from micro-organisms. It is preferred that the degree of homology is at least 80%, more preferred that the degree of homology is at least 85% and still more preferred that the degree of homology is at least 90%. It is most particularly preferred that the degree of homology is at least 95%.
The present invention includes also DNA sequences obtained by in vivo and in vitro recombination using SST and/or FFT-encoding sequences from plants and fructosyltransferase-encoding sequences from other prokaryotic or eukaryotic sources, including bacteria and fungi.
The present invention relates to SST-encoding DNA sequences from plants, which after insertion into the genome of a host organism induce the synthesis of oligofructans comprising of 2, 3 and/or 4 fructosyl units [G-(F).sub.n, 2.ltoreq.n.ltoreq.4]. The present invention also relates to FFT-encoding DNA sequences from plants which after insertion into the genome of a host organism together with SST-encoding DNA sequences, induce the synthesis of fructans with a higher degree of polymerization [G-(F).sub.n, n>4].
The present invention also relates to chimeric gene constructs comprising sequences encoding SST or FFT, or part of the sequences, the sequences being present in the antisense orientation. Introduction of these antisense constructs in plants which can synthesize fructans, will cause inhibition of SST or FFT catalysed reactions or cause inhibition of SST or FFT expression.
The present invention relates to chimeric gene constructs encoding SST, or part of the sequence, the coding sequence being present in antisense orientation. Introduction of these antisense constructs into the genome of host plants which can synthesize fructans, such as species of the Asteraceae, Liliaceae and Poaceae family, reduce or block the conversion of sucrose into oligofructans [G-(F).sub.n, 2.ltoreq.n.ltoreq.4]. Since only SST is able to catalyse the first step of fructan synthesis (reaction 2), in such transgenic plants, also the synthesis of fructans with a higher degree of polymerization, [G-(F).sub.n, n>4], will be reduced or blocked and these plants will accumulate sucrose rather than fructans.
The present invention relates to chimeric gene constructs encoding FFT, or part of the sequence, the coding sequence being present in the antisense orientation. Introduction of these antisense constructs into the genome of host plants which can synthesize fructans, reduce or block the conversion of oligofructans [G-(F).sub.n, 2.ltoreq.n.ltoreq.4] into fructans with a higher degree of polymerization [G-(F).sub.n, n>4]. Transgenic plants thus obtained will accumulate oligofructans, rather than fructans with a higher degree of polymerization.
Accordingly, the present invention provides a method for producing a genetically transformed host organism showing a modified fructan profile, which comprises the steps of:
i) preparing a chimeric gene construct comprising one or more DNA fragments as defined above, or said DNA fragments in the inverted orientation, operably linked to a promoter sequence active in said host organism and a terminator sequence active in said host organism,
ii) introducing the chimeric gene construct into the genome of the host organism.
The host organism may be a microorganism or a plant. In case the host organism is a plant, the method additionally comprises the step of
iii) regenerating the transformed plant cells to transgenic plants.
More specifically the method of the invention comprises the following steps:
a. construction of a chimeric gene comprising essentially the following sequences:
a promoter which ensures the formation of a functional RNA or protein in the intended target organism, target organs, tissues or cells,
a DNA sequence encoding SST or FFT,
a transcription terminator operationally connected to the DNA sequence, the SST or FFT-encoding DNA sequence being functionally connected to a promoter,
a DNA sequence encoding a targeting signal or a transit peptide which ensures targeting of SST or FFT to a specific subcellular compartment;
b. introduction of the chimeric gene into the genome of a host organism so as to obtain genetic material comprising the DNA sequence and
c. regeneration of the genetic material in a transformed host organism.
In the recombinant DNA of the present invention, the DNA sequence encoding SST or FFT is preferably linked to a regulatory sequence that ensures proper expression of the DNA sequence in a host organism, such as a bacterium, a yeast, an alga or a plant, at a sufficiently high expression level. Regulatory sequences are a promoter, a termination signal and a transcription or translational enhancer. A promoter can be the 35S promoter of the cauliflower mosaic virus (CaMV), or a sugar-inducible promoter like the patatine promoter or an organ-specific promoter like the tuber-specific potato proteinase inhibitor II promoter or any other inducible or tissue-specific promoter.
In the recombinant DNA of the present invention, the SST or FFT-encoding DNA sequence is preferably linked to regulatory sequences that are operative in plants and that ensure proper expression of the DNA sequence in the different plant organs, tissues or cells. A highly preferable promoter is a promoter which is active in organs and cell types which normally accumulate sucrose (the primary substrate for fructan synthesis). The production of fructans is particularly advantageous in organs storing large amounts of sucrose, such as the tap roots of sugar beet or the stems of sugar cane. Besides roots, other organs or cell types are involved in the synthesis, processing, transport and accumulation of sucrose. Therefore, SST or FFT-encoding sequences are also suitably expressed in leaves, stems, roots, tubers, reproductive organs, and seeds.
In the recombinant DNA of the present invention, the DNA sequence encoding SST or FFT contains, or is linked to, a sequence encoding a transit peptide which directs the SST or FFT mature protein to a subcellular compartment containing sucrose. The production of fructans is particularly advantageous in the vacuole which can accumulate very high concentrations of sucrose (up to 900 mol m.sup.-3). Besides the vacuole, other subcellular compartments are involved in the synthesis (cytoplasm), processing (cytoplasm, mitochondria, plastids) and transport (cell wall, cytoplasm) of sucrose. The present invention therefore relates to the use or sequences that allow targeting of the SST or FFT product to specific subcellular compartments, such as the vacuole, the cell wall, mitochondria, plastids and cytoplasm.
The present invention also relates to gene constructs, comprising a sequence encoding SST or FFT, or part of the sequences, the coding sequence being present in the antisense orientation. In these gene constructs, the SST or the FFT-encoding sequence is preferably linked to a promoter that ensures the formation of an antisense RNA in the cell types which normally were able to synthesize fructans.
The recombinant DNAs of the present invention may also encode proteins having herbicide resistance, plant growth promoting, plant growth inhibiting, anti-fungal, anti-bacterial, anti-viral and/or anti-nematode properties or conferring stress resistance. The recombinant DNAs of the present invention may further encode proteins which induce sterility. In case that the DNA is to be introduced into a heterologous organism it may be modified to remove known mRNA instability motifs (such as AT rich regions); and polyadenylation signals, and/or codons which are preferred by the organism, into which the recombinant DNA is to be inserted, are used so that expression of the thus modified DNA in the host organism is higher than that obtained by expression of the unmodified recombinant DNA in the same host organism.
The present invention also provides the transformed host organism, especially a transformed plant, produced by the above method. The invention preferably includes agricultural, forage, vegetable, ornamental and fruit crops, more preferably sugar beet, sugar cane, potato, petunia, alfalfa, soybean, rice, ryegrass, thimothygrass, wheat, barley, sorghum, maize, chicory, Jerusalem artichoke, tulip, melon, onion, garlic, tomato, strawberry, apple and pear. Moreover, the invention includes a plant cell, seed, fruit, seedling or any plant part harbouring recombinant DNA, comprising a sequence encoding SST or FFT as defined herein. Further, this invention includes the progeny of the transformed plants which contain the DNA stably incorporated and heritable in a Mendelian manner and/or the seeds of such plants and such progeny.
EXPERIMENTAL
Purification of 1-SST
Tubers of Helianthus tuberosus `Colombia` were used for the extraction of 1-SST and 1-FFT. Tubers were harvested in August-September, during the period of rapid tuber growth and massive fructan accumulation. Tubers were washed and frozen in liquid nitrogen. Four hundred gram of frozen tubers (-80.degree. C.) were pulverized and immediately thereafter homogenized in a Waring blender in 900 cm.sup.3 50 mol m.sup.-3 phosphate (P) buffer, pH 6.5, containing 10% (w/v) glycerol, 1 mol m.sup.-3 MgSO.sub.4, 1 mol m.sup.-3 Na.sub.2 EDTA, 1 mol m.sup.-3 PMSF (Sigma, USA) 1 mol m.sup.-3 DTT, 1.5% (w/v) PVPP and 20 mmol m.sup.-3 Na.sub.2 S.sub.2 O.sub.5. The homogenate was filtered through three layers of Miracloth and centrifuged at 17,000 g for 1 h.
The protein extract was kept at 4.degree. C. and adjusted to 45% saturation with (NH.sub.4).sub.2 SO.sub.4. The insoluble proteins were pelleted by centrifugation (10,000 g, 30 min) and discarded. The 45% supernatant was brought to 70% saturation by further addition of (NH.sub.4).sub.2 SO.sub.1. The pellet, obtained after a second centrifugation step, was redissolved in 60 cm.sup.3 50 mol m.sup.-3 phosphate (P) buffer, pH 6.5, 1 mol m.sup.-3 DTT and 1 mol m.sup.-3 PMSF (Sigma, USA), and desalted by dialysis against 10 mol m.sup.-3 P-buffer, pH 6.5, 1 mol m.sup.-3 PMSF and 1 mol m.sup.-3 DTT, for 16 h. After buffer replacement, dialysis was continued for another 3 h. The whole procedure was performed at temperatures between 0 and 4.degree. C. The centrifuged dialysate (30,000 g, 30 min) was applied onto a 25.times.120 mm Q Sepharose Phast Flow column (4.degree. C.), which had been prewashed with 10 mol m-3 bis Tris, pH 6.5, 1 mol m.sup.-3 DTT, 1 mol m.sup.-3 PMSF and 5 mol m.sup.-3 EDTA in Milli Q water. Bound proteins were eluted with an NaCl gradient (0-300 mol m.sup.-3) in the same buffer at a flow rate of 5 cm.sup.3 min.sup.-1. 1-SST eluted at 200-250 mol m.sup.-3 NaCl.
The Q Sepharose fractions were adjusted to 400 mol m.sup.-3 with solid (NH.sub.4).sub.2 SO.sub.4. Fractions of 20 cm.sup.3 were loaded onto a 15.times.50 mm column of Phenyl Sepharose High Performance or Phenyl Sepharose High Substitution, which where pre-equilibrated with 10 mol m.sup.-3 bis Tris buffer, pH 6.5, containing 500 mol m.sup.-3 (NH.sub.4).sub.2 SO.sub.4, 1 mol m.sup.-3 DTT, 1 mol m.sup.-3 PMSF, 2 mol m.sup.-3 EDTA and 0.1% CHAPS (buffer A) at 12.degree. C. Elution of bound proteins was carried out using a linear gradient of buffer A (100-0%) without (NH.sub.4).sub.2 SO.sub.4, containing 25% (v/v) ethylglycol, at a flow rate of 1 cm.sup.3 min.sup.-. 1-SST eluted at 100 mol m.sup.-3 (NH.sub.4).sub.2 SO.sub.4.
Phenyl Sepharose fractions up to 10 cm.sup.3 were injected onto a 5.times.50 mm Concanavalin A Sepharose column, prewashed in 20 mol m.sup.-3 bis-Tris, pH 6.5, 250 mol m.sup.-3 NaCl, 0.5 mol m.sup.-3 CaCl.sub.2, 0.5 mol m.sup.-3 MnCl.sub.2, 1 mol m.sup.-3 DTT and 1 mol m.sup.-3 PMSF. Bound 1-SST was eluted with 500 mol m.sup.-3 .alpha.-CH.sub.3 -mannopyranoside in the same buffer.
Active fractions of one Concanavalin A-run were pooled and applied to a 5.times.200 column packed with spherical (15 .mu.m) hydroxylapalite (Merck, Germany). The column was pre-equilibrated in 2 mol m.sup.-3 CaCl.sub.2, 10 mol m.sup.-3 NaCl, 1, mol m.sup.-3 DTT, 1 mol m.sup.-3 PMSF and 0.1% CHAPS (buffer A). Proteins bound to the column were eluted with stepped gradient of buffer A and 500 mol m.sup.-3 potassium phosphate buffer, pH 6.5, at a flow rate of 0.5 ml min.sup.-1. 1-SST eluted at 75-100 mol m.sup.-3 potassium phosphate.
Active fractions of one hydroxylapatite run were pooled and applied onto a 5.times.50 mm Mono Q-column which was pre-equilibrated with 10 mol m.sup.-3 P-buffer, pH 6.5, 1 mol m.sup.-3 DTT, 1 mol mu.sup.-3 EDTA and 0.1% CHAPS. Bound proteins were eluted with an NaCl gradient (0-500 mol m.sup.-3) at a flow rate of 0.5 cm.sup.3 min.sup.-1. 1-SST eluted at 250 mol m.sup.-3 NaCl.
All columns, column packings and chromatography equipment were obtained from Pharmacia (Sweden), unless indicated otherwise.
Purification of 1-FFT
A crude protein extract was obtained from tubers of H. tuberosus as described for the purification of 1-SST. The supernatant of the crude protein extract, as obtained after centrifugation was adjusted to 45% saturation with (NH.sub.4).sub.2 SO.sub.4 and stirred for 1 h. The insoluble proteins were pelleted by centrifugation at 10,000 g for 30 min. The pellet was redissolved in 60 cm.sup.3 50 mol m.sup.-3 P-buffer, pH 6 5, 1 mol m.sup.-3 DTT and 1 mol m.sup.-3 PMSF, and dialyzed overnight against 10 mol m.sup.-3 citrate/phosphate (C/P) buffer, pH 4.5, containing 1 mol m.sup.-3 PMSF and 1 mol m.sup.-3 DTT. The contents of the dialysis tubing was readjusted to pH 6.5 with 0.2 M Na.sub.2 HPO.sub.4 and the insoluble proteins were removed by centrifugation at 30.000 g for 1 h. The supernatant was loaded onto a 25.times.120 mm column of Q Sepharose Phast Flow (Pharmacia) pre-equilibrated with 10 mol m.sup.-3 P-buffer, pH 6.5, 1 mol m.sup.-3 DTT and 1 mol m.sup.-3 PMSF in Milli Q water ((Millipore B.V., The Netherlands). The column was cooled to 4.degree. C. Bound proteins were eluted with a positive NaCl gradient at a flow rate of 5 cm.sup.3 min.sup.-1. 1-FFT eluted at 200-250 mol m.sup.-3 NaCl.
Solid (NH.sub.4).sub.2 SO.sub.4 was added to the Q Sepharose fractions to give a final concentration of 750 mol m.sup.-3 with. Fractions of 5 cm.sup.3 were loaded onto a 15.times.50 mm column of Phenyl Sepharose High Performance (Pharmacia), pre-equilibrated with 10 mol m.sup.-3 P-buffer, pH 6.5, containing 750 mol m.sup.-3 (NH.sub.4).sub.2 SO.sub.4 and 1 mol m.sup.-3 DTT. Bound proteins were eluted with a negative (NH.sub.4).sub.2 SO.sub.4 gradient at a flow rate of 1 cm.sup.3 min.sup.-1 at 12.degree. C. 1-FFT eluted at 450-400 mol m.sup.-3 (NH.sub.4).sub.2 SO.sub.4.
Phenyl Sepharose fractions (up to 4 cm.sup.3) were injected onto a Hiload 16.times.600 mm Superdex 75 prep grade column (Pharmacia) pre-washed in 10 mol m.sup.-3 P-buffer, pH 6.5, and 1 mol m.sup.-3 DTT. Proteins were eluted in the same buffer at a flow rate of 0.5 cm.sup.3 min.sup.-1 at 20.degree. C. 1-FFT eluted 95-105 min after injection.
1-FFT and 1-SST assays
The 1-FFT activity of column fractions was routinely assayed at 35.degree. C. Aliquots of 25 mm.sup.3 were mixed with 25 mm.sup.3 0.3 g Neosugar P (Meiji Seika Kaisha, Ltd, Tokyo, Japan: Neosugar consists of 1% hexoses, 4% sucrose, 42% G-(F).sub.2, 44% G-(F).sub.3 and 7% G-(F).sub.4) per cm.sup.3 of 100 mol m.sup.-3 C/P-buffer, pH 6.5. After 3 h, the reaction was stopped by boiling the incubation mixture in a waterbath for 5 min. A net gain of GF.sub.4 was taken as a measure for 1-FFT activity.
For 1-SST activity, 15 mm.sup.3 column fractions were mixed with 15 mm.sup.3 500 mol m.sup.-3 GP in 100 mol m.sup.-3 C/P-buffer, pH 5.0 and incubated for 3 h at 35.degree. C. GF.sub.2 synthesis was taken as a measure for 1-SST activity.
Analysis of sugars and fructans
Sucrose and oligofructans were analyzed by RP-HPLC using a 2.1.times.220 mm Speri-5 RP 18 column (Brownlee Labs, Santa Clara, USA). Milli Q water was used as the eluant at a flow rate of 0.3 cm.sup.3 min.sup.-1 at 37.degree. C. Glucose and fructose were quantified on a 6.5.times.300 mm Shodex SC-1011 column (Millipore B.V., Waters Chromatography Division, The Netherlands) run at 85.degree. C. with Milli Q water at 0.75 cm.sup.3 min.sup.-1. Sugars were detected with a 2142 refraction index detector (RID, Pharmacia). Identification of oligofructans was by comparison of their retention times with those from the oligofructans purified from Neosugar P or from H. tuberosus and by the glucose/fructose ratios of the individual oligofructans (Koops and Jonker, 1994).
HPAEC analyses of oligofructans and fructans with a higher degree of polymerization were performed on a Dionex Series 4000 ion chromatograph equipped with 250.times.4 mm CarboPac PA1 anion exchange column and a 25.times.3 mm CarboPac PA guard column. Fructans were separated with a 60 min linear gradient of 0.25 to 0.4 mol m.sup.-3 NaAc in 0.1 mol m.sup.-3 NaOH at a flow rate of 1 ml min.sup.-1. Detection was by pulsed amperometry (PAD) with a gold-working electrode. The applied potential of a pulse was kept at 0.1, 0.6 and -0.6 V for 0.5, 0.1 and 0.05 seconds, respectively. Rhamnose was used as an internal standard. Fructans were identified by comparison of their retention times with those of fructan standards isolated and purified from H. tuberosus according to the method of Heinze and Praznik (1991).
Amino acid sequencing of 1-SST and 1-FFT
Mono Q fractions of 1-SST or Superdex 75 fractions of 1-FFT were desalted and concentrated by centrifugation in Centricon-10 (2 cm.sup.3) ultrafiltration devices (Grace B.V., Amicon division, The Netherlands) and subsequently collected by precipitation in 80% (v/v) aqueous acetone at -20.degree. C. 1-SST and 1-FFT (each 50 .mu.g) were dissolved in SDS-buffer (Laemmli, 1970) and separated on a pre-cast ExcelGel SDS, gradient 8-18. Proteins were stained with Coomassie Brilliant Blue according to Rosenfeld et al. (1992). The stained 1-SST (2 bands; due to its intrinsic lability 1-SST is cleaved by the SDS treatment) and 1-FFT bands were excised with a scalpel and washed, dried and partially rehydrated according to the procedure of Rosenfeld et al. (1992). Sequencing-grade trypsin (0.5 .mu.g; Boehringer, Germany) was added to the gel slice, and in-gel digestion of the proteins was carried out for 4 h at 30.degree. C. The resulting peptides were recovered by two extractions of 20 min each, with 50 .mu.l acetonitrile, water, trifluoracetic acid and Tween 20 (60:40:0.001:0.0002, v/v). The resulting peptide mixture was separated by preparative RP-HPLC on a 9.3.times.250 mm SuperPac Pep-S column (Pharmacia) eluted with a linear gradient of 0.1% TFA in 0 to 60% aqueous acetonitrile at a flow rate of 4 ml min.sup.-1. Individual peptide fractions were collected manually and stored at -80.degree. C. The amino acid sequences of selected peptides were determined by Edman degradation, using a model 417A pulse-liquid sequenator, connected on-line to a model 120A RP-HPLC unit (Applied Biosystems). Amino acid sequences specific for 1-SST or FTT were translated into the corresponding degenerated DNA sequences (Example 1, Table 1), which, in turn, were used as primers for ACR.
DNA methodology
DNA and RNA isolation, subcloning, restriction analysis and sequencing were performed using standard methods described in molecular biology manuals (Sambrook et al. 1989, Ausubel et al. 1994).
cDNA synthesis
Poly (A).sup.+ RNA was isolated from tubers of H. tuberosus `Colombia`. Single stranded cDNA was synthesized by reverse transcriptase from 10 .mu.g poly(A).sup.+ RNA by priming the poly(A).sup.+ RNA with the following tail specific primer: 5'-CCGAATTCAATACGACTCACTATAGCG(T).sub.15 -3'
PCR
Degenerated oligonucleotides specific for 1-SST or 1-FFT and the tail specific primer, 5'-CCGAATTCAATACGACTCACTATAGCG-3' were used for amplification of the single strand cDNA. PCR was performed in 50 .mu.l PCR buffer (Life Technologies), containing 100 pmol cDNA template, 100 pmol of the tail specific primer and 100 pmol of primers specific for 1-SST or 1-FFT. Amplification involved 30 cycles of denaturing (1 min, 92.degree. C.), annealing (1 min, 42.degree. C.) and amplification (1 min, 72.degree. C.). The resulting fragments were electrophoresed in 0.7% agarose, excised from the gel, isolated from the agarose matrix and subcloned into pMOSBlue vector (Amersham). 1-SST and 1-FFT specific fragments generated by PCR were used to screen a Uni-ZAP XR cDNA library.
Construction and screening of a cDNA library
Ten .mu.g of poly(A).sup.+ RNA isolated from tubers of H. tuberosus `Colombia` was used as starting material for the construction of an Uni-ZAP XR cDNA library (Stratagene). Construction, plating and screening of the library were performed according to the protocols developed by Stratagene (La Jolla, Calif., cat. no. 237211). .sup.32 P-labelled DNA probes specific for 1-SST or 1-FFT were prepared by random oligonucleotide priming and used to screen about 100.000 plaques. Hybridization and washing of Hybond-N membrane were performed under high stringency conditions (hybridization at 65.degree. C., final wash step with 0.1.times.SSC, 0.1% SDS, 65.degree. C.). Positive clones were purified, the pbluescript phagemids excised from the Uni-ZAP vector using the Exassist/Solr system (Stratagene) and the inserts by restriction enzyme analysis, hybridization and sequencing.
Analysis of transgenic plants
Analysis of sugars and fructans
50 mg of leaf tissue was frozen in liquid N.sub.2 and homogenized in 0.1 cm.sup.3 Milli Q water in an Eppendorf tube. The homogenate was heated to 90.degree. C. for 5 min, then centrifuged at 14,000 g for 10 min. The clear supernatant was analysed by TLC: 2 .mu.l of the supernatant was spotted onto a silica gel G 1500 (Schleicher & Schuell) plate. TLC plates were developed two times with either acetone, water (9:1, v/v), or butan-1-ol, propan-2-ol and water (3:12:4, v/v). Carbohydrates were visualized by spraying the TLC plates with urea-phosphoric acid (Wise et al. 1955).
1-FFT assay
Leaf tissue (50 mg) was frozen in liquid N.sub.3 and homogenized in an Eppendorf tube in 0.1 cm.sup.3 25 mol m.sup.-3 phosphate (P) buffer, pH 6.5, containing, 2 mol m.sup.-3 MgSO.sub.4, 2 mol m.sup.-3 Na.sub.3 EDTA, 2 mol m.sup.-3 PMSF (Sigma, USA) 2 mol m.sup.-3 DTT, 1.5% (w/v) soluble PVPP (Merck) and 20 mol m.sup.-3 Na.sub.2 S.sub.2 O.sub.5. The homogenate was centrifuged at 14,000 g for 10 min at 4.degree. C. The clear supernatant was used for the 1-FFT assay. Fifty .mu.l of the supernatant was mixed with 50 .mu.l of an assay mixture, containing 2 mol m-.sup.-3 G-(F).sub.3, 80 mol m.sup.-3 G-(F).sub.4, 50 mol m.sup.-3 citrate/phosphate buffer, pH 5.5, and 0.02% (w/v) NaN.sub.3. The assay mixture was incubated in the dark at 28.degree. C. Samples of 15 .mu.l were taken after 4, 20, 44 and 68 h of incubation, and analysed by TLC.
EXAMPLES
Example 1
Purification of the sucrose:sucrose fructosyltransterase (1-SST) and fructan fructosyltransferase (1-FFT), and isolation of 1-SST and 1-FFT- encoding cDNAs
1-SST and 1-FFT were purified from tubers of Helianthus tuberosus using precipitation techniques, several successive chromatography procedures and electrophoresis. Fractions with 1-SST-activity, eluting from the Mono Q column gave one band after native PAGE. 1-SST is cleaved by SDS, therefore analysis by SDS PAGE yielded two bands of 27 and 55 kDa (FIG. 1, lane 2). Fractions with 1-FFT-activity eluting from the Superdex 75 gel permeation column gave on SDS PAGE one band with an estimated molecular weight of 70 kDa (FIG. 1, lane 3). 1-SST alone was able to synthesize oligofructans from sucrose as a sole substrate (FIG. 3A, 80 h of incubation). By recombining purified 1-SST with purified 1-FFT, sucrose could be converted into fructans with a deree of polymerization of at least 15 [G-(F).sub.14, FIG. 3B, 80 h of incubation].
For amino acid sequencing by the Edman (phenylisothiocyanate) degradation method, the 27 kDa (1-SST), 55 kDa (1-SST) and 70 kDa (1-FFT) protein bands were excised from the SDS PAGE gel and subjected to proteolytic digestion by trypsin. The resulting peptide mixtures were separated by RP-HPLC in separate runs (FIGS. 2A-C). The peptides eluting after 26 min and 37 min (both from the 25 kDa fragment of 1-SST, FIG. 2A), 27 min, 34 min and 37 min (from the 55 kDa fragment of 1-SST, FIG. 2B), 28 min, and 32 min (from 1-FFT, FIG. 2C, the fraction eluting at 32 min contained the peptides 7 and 8) were collected manually and subjected to N-terminal amino acid sequencing (Table 1).
TABLE 1__________________________________________________________________________Amino acid sequences of se1ected peptides obtained after tryptic digestion of 1-SST (25 and 55 kDa polypeptides) and 1-FFT, and separation of the resulting peptide mixtures by RP-HPLCprotein amino acid sequence DNA sequence of PCR-primer__________________________________________________________________________ 1-SST,25 kDa ADVLF??TTSEGSVAR - 2 idem EQLPVYFYIAK 5'-GARCARYTNCCNGTNTAYTTYTAYATH GCNAAR-3' - 3 1-SST,55 kDa VVLDLETK - 4 idem FRDPSTLWL?PDGEY - 5 idem GWANIL - 6 1-FFT GWATVYNVGR 5'-GGNTGGGCNACNGTNTAYAAY-3' - 7 idem LLVDHSIVEGFAQGGR 5'-ATHGTNGARGGN TTYGCNCAR-3' - 8 idem VGESDS__________________________________________________________________________
Amino acid sequences 2 (1-SST, 25 kDa), 6 and 7 (1-FFT) were used to design DNA primers specific for 1-SST or 1-FFT which were used for PCR. PCR using primers 2, 6 or 7 yielded DNA of about 450, 600 bp and 300 bp, respectively. Nested PCR, using oligonucleotide 7 as specific primer and the 800 bp PCR fragment as template, again yielded the 300 bp PCR fragment which indicated that the 300 bp fragment is included in the 800 bp fragment.
An Uni-ZAP cDNA library constructed from mRNA isolated from H. tuberosus tubers was screened with either the 450 bp 1-SST or the 800 bp 1-FFT fragment. Screening of about 100.000 cDNA clones yielded about 20 positive clones hybridizing to the 450 bp fragment and 25 clones hybridizing to the 800 bp fragment. DNA of clones hybridizing to the 450 bp fragment did not hybridize to the 800 bp fragment and vice versa. Positive clones were purified, the pBluescript phagemids excised from the uni-ZAP vector and the insert characterized by restriction enzyme analysis, hybridization and sequencing.
The DNA sequences of 1-SST and 1-FFT and their corresponding amino acid sequences are presented in FIG. 4A and FIG. 4B, respectively. Sequence ID. No. 1, encoding 1-SST, has an open reading frame of 1890 base pairs and encodes a protein of 630 amino acid residues. On DNA level, 1-SST shows a 68% identity with soluble acid .beta.-fructofuranosidase (=acid invertase) cDNA from carrot (Daucus carota). At the amino acid level, 1-SST shows a 66% similarity with soluble acid .beta.-fructofuranosidase from carrot.
Sequence ID. No. 3, encoding 1-FFT, has an open reading frame of 1845 base pairs and encodes a protein of 615 amino acid residues and a molecular weight of about 69 kDa. This corresponds to the molecular weight of the purified 1-FFT protein as established by SDS-ACAGE (Koops and Jonker, 1994). On DNA level, 1-FFT shows a 65% identity with soluble acid .beta.-fructofuranosidase (=acid invertase) cDNA from carrot. At the amino acid level, 1-FFT shows a 60% similarity with soluble acid .beta.-fructofuranosidase from carrot.
Although 1-SST and 1-FFT have a relatively high degree of homology with acid invertase it has been shown that 1-SST or 1-FFT and invertase are distinctly different enzymes. It has been shown that 1-SST and 1-FFT are unable to catalyse the hydrolysis of sucrose (invertase activity). Hydrolytic activity of purified 1-SST and 1-FFT against sucrose has been tested at a range of pH's and sucrose concentrations. There was no significant invertase activity under any of these conditions (Koops and Jonker 1994). No homology higher than 68% was observed between the DNA sequence encoding 1-SST and any known DNA sequence within the PDB, GENBANK, GENBANK updates, EMBL and EMBL updates nucleotide sequence databases. No homology higher than 65% was observed for the DNA sequence encoding 1-FFT with any known DNA sequence within the PDB, GENBANK, GENBANK updates, EMBL and EMBL updates nucleotide sequence databases.
Example 2
Construction of a chimeric sst gene
The full length sst cDNA clone, designated pSST 103, was used for the introduction of an NcoI site at the ATG (position 34), and a EcoRV site downstream of the stopcodon (at position 1924) using PCR. From the plasmid pMOG18 (Pen et al., 1992) which contains the enhanced CaMV35S promoter, ALMV leader sequence, uidA gene and the nos terminator sequence, the uidA coding sequence was replaced by the sst cDNA. pMOGl8 was digested with BamHI, filled in with Klenow DNA polymerase, and digested with NcoI. The sst PCR fragment, cut with NcoI and EcoRI, was ligated into this vector, resulting in the clone pSST217. The EcoRI/HindII fragment of pSST217 containing the complete chimeric construct (enh.35S+ALMV-sst-nos) was cloned into the EcoRI and HindIII site of pBINPLUS (Van Engelen et al., 1995), a binary plant transformation vector derived from pBINl9 (Bevan, 1984) resulting in plasmid pVSl (FIG. 5A).
Example 3
Construction of a chimeric fft gene
The full length fft cDNA clone, designated pFFT 111, was used for the introduction of an NcoI site at the ATG (position 29), and a BamHI site downstream of the stopcodon (at position 1874) using PCR. From the plasmid pMOG18 (Pen et al., 1992) which contains the enhanced CaMV35S promoter, ALMV leader sequence, uidA gene and the nos terminator sequence, the uidA coding sequence was replaced by the fft cDNA. The PCR fragment digested with NcoI and BamHI was ligated into the pMOG18 vector digested with NcoI and BamHI, resulting in clone pFFT209. The HindIII/EcoRI fragment of pFFT209 containing the complete chimeric construct (enh.35S+ALMV-fft-nos) was cloned into the HindIII and EcoRI site of pBINPLUS (Van Engelen et al., 1995), a binary plant transformation vector derived from pBIN19 (Bevan, 1984) resulting in plasmid pVF1 (FIG. 5B).
Example 4
Transformation of Petunia and potato plants
The binary vectors pVS1 and pVF1 (FIG. 5) were conjugated from E. coli XL1-Blue to Agrobacterium tumefaciens strain AGLO by triparental mating (Ditta et al., 1980). Exconjugants were used to transform Petunia hybrida leaf discs as described by Horsch et al. (1985). Leaf discs were prepared from top leaves of young non-flowering plants. P. hybrida variety W115 was used for the transformation experiments. Exconjugants were also used to transform diploid potato (Solanum tuberosum, variety Kardal) stem explants as described previously (Visser, 1991). After shoot and root regeneration on kanamycin-containing media, plants were put in soil and transferred to the greenhouse. Plants regenerated (on kanamycin-free media) from leaf discs and stem explants treated with the Agrobacterium strain AGLO lacking a binary vector served as a control.
Example 5
Analysis of transgenic plants expressing the sst and fft genes
About 25 transgenic petunia plants and 25 transgenic potato plants were generated harboring the pVS1 construct and 25 transgenic petunia and potato plants harboring the pVFl construct. Ten petunia and ten potato plants were transformed with the Agrobacterium strain AGL0 lacking a binary vector. These plants were used as a control. Southern blot analysis of genomic DNA isolated from the transformed plants showed that on average 1-5 copies of the introduced chimeric genes were integrated (data not shown).
The carbohydrate composition of transgenic plants was analysed by two essentially different techniques: thin layer chromatography (TLC), which separate carbohydrates on basis of liquid--liquid partitioning (after TLC, the fructans were detected by a fructose specific colour reaction) and HPAEC, which separate carbohydrates at alkaline conditions (pH 13) on basis of charge, and detect carbohydrates by oxidation with a gold working electrode. Analysis of leaves extracts from the potato and Petunia plants harboring the pVS1 construct showed that both transgenic plant species contain products which are the result of SST activity. TLC showed the presence of at least the trisaccharide G-(F).sub.2 and most probably also the tetrasaccharide G-(F).sub.3 and the pentasaccharide G-(F).sub.4 in extracts of potato leaves, whereas these oligofructans were absent in the control plant (FIG. 6). The presence of G-(F).sub.2 and G-(F).sub.3 in leave extracts of potato but also of transgenic P. hybrida plants was demonstrated by HPAE analysis (FIGS. 7 and 8). HPAEC, which is more sensitive and more specific than TLC, also revealed a small amount of G-(F).sub.4 in transgenic potato (FIG. 8). The results of FIGS. 6-8 clearly indicate that the sst gene is expressed into an enzymatically active SST protein in both P. hybrida and potato.
Transgenic plants harboring the pVF1 construct did not contain fructans because FFT needs oligofructans (such as G-(F).sub.2 or G-(F).sub.3) as initial substrates for the synthesis of fructans. Oligofructans are not present in plants lacking SST activity such as wild type potato or Petunia, or transgenic plants only containing the pVF1 construct. The presence of FFT activity in transgenic plants harboring the pVF1 construct is therefore verified by FFT activity measurements. The FFT assay used to evaluate the presence of an active FFT in transgenic potato was based on the ability of FFT to catalyse the synthesis of G-(F).sub.n, n>4, at the expense of G-(F).sub.4. The total leaf protein extract from potato plants harboring the pVF1 construct, was mixed with an G-(F).sub.4 containing assay mixture, and the presence of fructans with a higher degree of polymerization (G-(F).sub.n, n>4) was examined by TLC. Already after 4 h of incubation, G-(F).sub.5 and G-(F).sub.6 could be detected. After 44 h of incubation, fructans with a degree of polymerization higher then 4, including G-(F).sub.4, G-(F).sub.5, G-(F).sub.6, G-(F).sub.7 and G-(F).sub.8, could be detected, whereas these fructans were absent in the control mixtures (FIG. 9). This indicates that also the fft gene is expressed into an enzymatically active FFT protein.
DEFINITIONS AND ABBREVIATIONS
Fructan nomenclature is as according to Lewis (1993)
Fructosyl unit: a fructose molecule linked to another sugar molecule (e.g. glucose, fructose or galactose). Abbreviated as -F (e.g. in G-F) or -F-(e.g. G-F-F) . For molecules consisting of more then one fructosyl unit (e.g. G-F-F-F-F-F) a condensed notation is used [G-(F).sub.6 ] or GF.sub.6. G-(F).sub.6 consists of one glucosyl and 6 fructosyl units. G-(F).sub.6 consists of one fructosyl-glucose linkage and 5 fructosyl-fructose linkages.
Oligofructans: Any compound with one, two, or three fructosyl-fructose linkages (a glucose may be present but is not necessary). In the present application, the term oligofructans was used to denote the products of SST-activity [G-(F).sub.2, G-(F).sub.3 and G-(F).sub.4 ]. Oligofructans are more generally denoted as short chain fructans or fructans with a low degree of polymerization. In the present application oligofructans also include compounds consisting of 2, 3 or 4 fructosyl units, but lacking the glucosyl unit.
Fructans: Any compound in which one or more fructosyl-fructose linkages constitute a majority of the linkages (a glucose may be present but is not necessary). Fructan is used as a numerative noun where the materials referred to are chemically distinct (e.g. the fructans G-(F).sub.2 and G-(F).sub.13. In the present application, fructans are defined as the products of FFT activity (G-F.sub.n, 2.ltoreq.n<.+-.60). Unless indicated otherwise, fructans include also oligofructans. To denote a restricted group of fructans the "G-F.sub.n, n= . . . " notation is used. In the present application fructans also include compounds consisting of more than 1 fructosyl unit, but lacking the glucosyl unit.
Inulin: Fructan that has mostly the .beta.-2,1-fructosyl-fructose linkage (a glucose may be present, but not is necessary). Cumulative vs. numerative usage as a noun is the same as for fructan above.
Levan: Fructan that has mostly the .beta.-2,1-fructosyl-fructose linkage (a glucose is allowed but not necessary). Levan is also used to denote fructans from bacterial origin, although bacterial fructans do not always consists of predominantly .beta.-2,6 fructosyl-fructose linkages. For example, the fructans synthesized by levansucrase from Bacillus subtilus have predominantly .beta.-2,1-fructosyl-fructose linkages (inulin). Cumulative vs. numerative usage of levan as a noun is the same as for fructan.
Levansucrase: Enzymes from bacterial origin that are involved in the synthesis of fructan.
Sucrose:sucrose fructosyltransferase (SST): Plant-derived enzyme catalyzing the initial step of fructan synthesis (reaction 2). Enzyme can also be involved in the synthesis of oligofructans [G-(F).sub.n, 2.ltoreq.n.ltoreq.4] (reaction 3). In the present application, the designation SST can include either 1-SST, an SST form involved in the biosynthesis of oligofructans that has mostly the .beta.-2,1-fructosyl-fructose linkage; or 6-SST, an SST form involved in the biosynthesis of oligofructans that has mostly the .beta.-2,6-fructosyl-fructose linkage; or 1-SST and 6-SST.
Fructan:fructan fructosyltransferase (FFT). Plant-derived enzyme involved in the synthesis of fructans. Enzyme capable of catalyzing the synthesis of oligofructans and fructans of a higher degree of polymerization. FFT from H. tuberosus has overlapping activity with SST from H. tuberosus (reaction 3), but cannot catalyse the initial step of fructan synthesis (reaction 2). In the present application, the designation FFT can include either 1-FFT, an FFT form involved in the biosynthesis of oligofructans that has mostly the .beta.-2,1-fructosyl-fructose linkage; or 6-FFT, an FFT form involved in the biosynthesis of fructans that has mostly the .beta.-2,6-fructosyl-fructose linkage; or 1-FFT and 6-FFT.
Invertase: .beta.-fructosidase or .beta.-fructofuranosidase.
Degree of polymerization (DP): term to indicate the total amount of fructosyl and glycosyl residues; for example, G-(F).sub.2 has a DP of 3. The n-value in G-F.sub.n increases with an increasing degree of polymerization.
Fructan profile: term to describe the fructan size/distribution pattern, or alternatively, the relative amounts and kinds of fructans, in an extract for example derived from a plant, plant organ or plant cell. The currently most reliable method to analyze a fructran pattern of an extract is by high performance anion exchange chromatography and pulsed amperometric detection (Chatterton et al. 1990).
Helianthus tuberosus: Jerusalem artichoke.
Cichorium intybus: chicory.
HPLC: High Performance Liquid Chromatography. Technique for the separation of complex mixtures of compounds. Variants on this technique are high performance reversed phase chromatography (RP HPLC) or high performance anion exchange chromatography in combination with pulsed amperometric detection (HPAEC-PAD, see for example FIG. 3).
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__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 16 - - - - (2) INFORMATION FOR SEQ ID NO: 1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2116 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pSST103 - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:34..1923 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 1: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 1: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1: - - GGCACGAGAA AAAACCCTCC CTCAGGCCAC CAC ATG ATG GCT TC - #A TCC ACCACC 54 - # - # Met Met Ala Ser Ser Thr Thr - # - # 1 - #5 - - ACC ACC CCT CTC ATT CTC CAT GAT GAC CCT GA - #A AAC CTC CCA GAA CTC 102 Thr Thr Pro Leu Ile Leu His Asp Asp Pro Gl - #u Asn Leu Pro Glu Leu 10 - # 15 - # 20 - - ACC GGT TCT CCG ACA ACT CGT CGT CTA TCC AT - #C GCA AAA GTG CTT TCG 150 Thr Gly Ser Pro Thr Thr Arg Arg Leu Ser Il - #e Ala Lys Val Leu Ser 25 - # 30 - # 35 - - GGG ATC CTT GTT TCG GTT CTG GTT ATA GGT GC - #T CTT GTT GCT TTA ATC 198 Gly Ile Leu Val Ser Val Leu Val Ile Gly Al - #a Leu Val Ala Leu Ile 40 - # 45 - # 50 - # 55 - - AAC AAC CAA ACA TAT GAA TCC CCC TCG GCC AC - #C ACA TTC GTA ACT CAG 246 Asn Asn Gln Thr Tyr Glu Ser Pro Ser Ala Th - #r Thr Phe Val Thr Gln 60 - # 65 - # 70 - - TTG CCA AAT ATT GAT CTG AAG CGG GTT CCA GG - #A AAG TTG GAT TCG AGT 294 Leu Pro Asn Ile Asp Leu Lys Arg Val Pro Gl - #y Lys Leu Asp Ser Ser 75 - # 80 - # 85 - - GCT GAG GTT GAA TGG CAA CGA TCC ACT TAT CA - #T TTT CAA CCC GAC AAA 342 Ala Glu Val Glu Trp Gln Arg Ser Thr Tyr Hi - #s Phe Gln Pro Asp Lys 90 - # 95 - # 100 - - AAT TTC ATT AGC GAT CCT GAT GGC CCA ATG TA - #T CAC ATG GGA TGG TAT 390 Asn Phe Ile Ser Asp Pro Asp Gly Pro Met Ty - #r His Met Gly Trp Tyr 105 - # 110 - # 115 - - CAT CTA TTT TAT CAG TAC AAC CCT CAA TCT GC - #C ATC TGG GGC AAC ATC 438 His Leu Phe Tyr Gln Tyr Asn Pro Gln Ser Al - #a Ile Trp Gly Asn Ile 120 1 - #25 1 - #30 1 -#35 - - ACA TGG GGC CAC TCG GTA TCG AAA GAC ATG AT - #C AAC TGG TTC CATCTC 486 Thr Trp Gly His Ser Val Ser Lys Asp Met Il - #e Asn Trp Phe His Leu 140 - # 145 - # 150 - - CCT TTC GCC ATG GTT CCT GAC CAT TGG TAC GA - #C ATC GAA GGT GTC ATG 534 Pro Phe Ala Met Val Pro Asp His Trp Tyr As - #p Ile Glu Gly Val Met 155 - # 160 - # 165 - - ACG GGT TCG GCT ACA GTC CTC CCT AAT GGT CA - #A ATC ATC ATG CTT TAC 582 Thr Gly Ser Ala Thr Val Leu Pro Asn Gly Gl - #n Ile Ile Met Leu Tyr 170 - # 175 - # 180 - - TCG GGC AAC GCG TAT GAT CTC TCC CAA GTA CA - #A TGC TTG GCG TAC GCT 630 Ser Gly Asn Ala Tyr Asp Leu Ser Gln Val Gl - #n Cys Leu Ala Tyr Ala 185 - # 190 - # 195 - - GTC AAC TCG TCG GAT CCA CTT CTT ATA GAG TG - #G AAA AAA TAT GAA GGT 678 Val Asn Ser Ser Asp Pro Leu Leu Ile Glu Tr - #p Lys Lys Tyr Glu Gly 200 2 - #05 2 - #10 2 -#15 - - AAC CCT GTC TTA CTC CCA CCA CCA GGA GTA GG - #C TAC AAG GAC TTTCGG 726 Asn Pro Val Leu Leu Pro Pro Pro Gly Val Gl - #y Tyr Lys Asp Phe Arg 220 - # 225 - # 230 - - GAC CCA TCC ACA TTG TGG TCG GGC CCT GAT GG - #T GAA TAT AGA ATG GTA 774 Asp Pro Ser Thr Leu Trp Ser Gly Pro Asp Gl - #y Glu Tyr Arg Met Val 235 - # 240 - # 245 - - ATG GGG TCC AAG CAC AAC GAG ACT ATT GGC TG - #T GCT TTG ATT TAC CAT 822 Met Gly Ser Lys His Asn Glu Thr Ile Gly Cy - #s Ala Leu Ile Tyr His 250 - # 255 - # 260 - - ACC ACT AAT TTT ACG CAT TTT GAA TTG AAA GA - #G GAG GTG CTT CAT GCA 870 Thr Thr Asn Phe Thr His Phe Glu Leu Lys Gl - #u Glu Val Leu His Ala 265 - # 270 - # 275 - - GTC CCA CAT ACT GGT ATG TGG GAA TGT GTT GA - #T CTT TAC CCG GTG TCC 918 Val Pro His Thr Gly Met Trp Glu Cys Val As - #p Leu Tyr Pro Val Ser 280 2 - #85 2 - #90 2 -#95 - - ACC GTA CAC ACA AAC GGG CTG GAC ATG GTG GA - #T AAC GGG CCA AATGTT 966 Thr Val His Thr Asn Gly Leu Asp Met Val As - #p Asn Gly Pro Asn Val 300 - # 305 - # 310 - - AAG TAC GTG TTG AAA CAA AGT GGG GAT GAA GA - #T CGC CAT GAT TGG TAT 1014 Lys Tyr Val Leu Lys Gln Ser Gly Asp Glu As - #p Arg His Asp Trp Tyr 315 - # 320 - # 325 - - GCA ATT GGA AGT TAC GAT ATA GTG AAT GAT AA - #G TGG TAC CCA GAT GAC 1062 Ala Ile Gly Ser Tyr Asp Ile Val Asn Asp Ly - #s Trp Tyr Pro Asp Asp 330 - # 335 - # 340 - - CCG GAA AAT GAT GTG GGT ATC GGA TTA AGA TA - #T GAT TTT GGA AAA TTT 1110 Pro Glu Asn Asp Val Gly Ile Gly Leu Arg Ty - #r Asp Phe Gly Lys Phe 345 - # 350 - # 355 - - TAT GCG TCC AAG ACG TTT TAT GAC CAA CAT AA - #G AAG AGG AGA GTC CTT 1158 Tyr Ala Ser Lys Thr Phe Tyr Asp Gln His Ly - #s Lys Arg Arg Val Leu 360 3 - #65 3 - #70 3 -#75 - - TGG GGC TAT GTT GGA GAA ACC GAT CCC CAA AA - #G TAT GAC CTT TCAAAG 1206 Trp Gly Tyr Val Gly Glu Thr Asp Pro Gln Ly - #s Tyr Asp Leu Ser Lys 380 - # 385 - # 390 - - GGA TGG GCT AAC ATT TTG AAT ATT CCA AGG AC - #C GTC GTT TTG GAC CTC 1254 Gly Trp Ala Asn Ile Leu Asn Ile Pro Arg Th - #r Val Val Leu Asp Leu 395 - # 400 - # 405 - - GAA ACT AAA ACC AAT TTG ATT CAA TGG CCA AT - #C GAG GAA ACC GAA AAC 1302 Glu Thr Lys Thr Asn Leu Ile Gln Trp Pro Il - #e Glu Glu Thr Glu Asn 410 - # 415 - # 420 - - CTT AGG TCG AAA AAG TAT GAT GAA TTT AAA GA - #C GTC GAG CTT CGA CCC 1350 Leu Arg Ser Lys Lys Tyr Asp Glu Phe Lys As - #p Val Glu Leu Arg Pro 425 - # 430 - # 435 - - GGG GCA CTC GTT CCC CTT GAG ATA GGC ACA GC - #C ACA CAG TTG GAT ATA 1398 Gly Ala Leu Val Pro Leu Glu Ile Gly Thr Al - #a Thr Gln Leu Asp Ile 440 4 - #45 4 - #50 4 -#55 - - GTT GCG ACA TTC GAA ATC GAC CAA AAG ATG TT - #G GAA TCA ACG CTAGAG 1446 Val Ala Thr Phe Glu Ile Asp Gln Lys Met Le - #u Glu Ser Thr Leu Glu 460 - # 465 - # 470 - - GCC GAT GTT CTA TTC AAT TGC ACG ACT AGT GA - #A GGC TCG GTT GCA AGG 1494 Ala Asp Val Leu Phe Asn Cys Thr Thr Ser Gl - #u Gly Ser Val Ala Arg 475 - # 480 - # 485 - - AGT GTG TTG GGA CCG TTT GGT GTG GTG GTT CT - #A GCC GAT GCC CAG CGC 1542 Ser Val Leu Gly Pro Phe Gly Val Val Val Le - #u Ala Asp Ala Gln Arg 490 - # 495 - # 500 - - TCC GAA CAA CTT CCT GTA TAC TTC TAT ATC GC - #A AAA GAT ATT GAT GGA 1590 Ser Glu Gln Leu Pro Val Tyr Phe Tyr Ile Al - #a Lys Asp Ile Asp Gly 505 - # 510 - # 515 - - ACC TCA CGA ACT TAT TTT TGT GCC GAC GAA AC - #A AGA TCA TCC AAG GAT 1638 Thr Ser Arg Thr Tyr Phe Cys Ala Asp Glu Th - #r Arg Ser Ser Lys Asp 520 5 - #25 5 - #30 5 -#35 - - GTA AGC GTA GGG AAA TGG GTG TAC GGA AGC AG - #T GTT CCT GTC CTCCCA 1686 Val Ser Val Gly Lys Trp Val Tyr Gly Ser Se - #r Val Pro Val Leu Pro 540 - # 545 - # 550 - - GGC GAA AAG TAC AAT ATG AGG TTA TTG GTG GA - #T CAT TCG ATA GTA GAG 1734 Gly Glu Lys Tyr Asn Met Arg Leu Leu Val As - #p His Ser Ile Val Glu 555 - # 560 - # 565 - - GGA TTT GCA CAA AAC GGG AGA ACC GTG GTG AC - #A TCA AGA GTG TAT CCA 1782 Gly Phe Ala Gln Asn Gly Arg Thr Val Val Th - #r Ser Arg Val Tyr Pro 570 - # 575 - # 580 - - ACA AAG GCG ATC TAC AAC GCT GCG AAG GTG TT - #T TTG TTC AAC AAC GCG 1830 Thr Lys Ala Ile Tyr Asn Ala Ala Lys Val Ph - #e Leu Phe Asn Asn Ala 585 - # 590 - # 595 - - ACT GGA ATC AGT GTG AAG GCG TCG ATC AAG AT - #C TGG AAG ATG GGG GAA 1878 Thr Gly Ile Ser Val Lys Ala Ser Ile Lys Il - #e Trp Lys Met Gly Glu 600 6 - #05 6 - #10 6 -#15 - - GCA GAA CTC AAT CCT TTC CCT CTT CCT GGG TG - #G ACT TTC GAA CTT 1923 Ala Glu Leu Asn Pro Phe Pro Leu Pro Gly Tr - #p Thr Phe Glu Leu 620 - # 625 - # 630 - - TGATGGTTAT ATTTTGGACC CTATATATGT GTTATTATCA TGATGGTTAT AT -#TTTGGACC 1983 - - CTATATATGT GTTATTATCA TGAAGCATAA GTTTGGACTG GAGGGGGTAT TA -#TTGTAATT 2043 - - TTATATGCAT GTTCTATTAC TTGTGAGGTT ATAGTATGTA ATTAAATTAT TA -#TATACTAT 2103 - - ATCAATTTCT AAT - # - # - # 2116 - - - - (2) INFORMATION FOR SEQ ID NO: 2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 630 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2: - - Met Met Ala Ser Ser Thr Thr Thr Thr Pro Le - #u Ile Leu His Asp Asp 1 5 - # 10 - # 15 - - Pro Glu Asn Leu Pro Glu Leu Thr Gly Ser Pr - #o Thr Thr Arg Arg Leu 20 - # 25 - # 30 - - Ser Ile Ala Lys Val Leu Ser Gly Ile Leu Va - #l Ser Val Leu Val Ile 35 - # 40 - # 45 - - Gly Ala Leu Val Ala Leu Ile Asn Asn Gln Th - #r Tyr Glu Ser Pro Ser 50 - # 55 - # 60 - - Ala Thr Thr Phe Val Thr Gln Leu Pro Asn Il - #e Asp Leu Lys Arg Val 65 - # 70 - # 75 - # 80 - - Pro Gly Lys Leu Asp Ser Ser Ala Glu Val Gl - #u Trp Gln Arg Ser Thr 85 - # 90 - # 95 - - Tyr His Phe Gln Pro Asp Lys Asn Phe Ile Se - #r Asp Pro Asp Gly Pro 100 - # 105 - # 110 - - Met Tyr His Met Gly Trp Tyr His Leu Phe Ty - #r Gln Tyr Asn Pro Gln 115 - # 120 - # 125 - - Ser Ala Ile Trp Gly Asn Ile Thr Trp Gly Hi - #s Ser Val Ser Lys Asp 130 - # 135 - # 140 - - Met Ile Asn Trp Phe His Leu Pro Phe Ala Me - #t Val Pro Asp His Trp 145 1 - #50 1 - #55 1 -#60 - - Tyr Asp Ile Glu Gly Val Met Thr Gly Ser Al - #a Thr Val Leu ProAsn 165 - # 170 - # 175 - - Gly Gln Ile Ile Met Leu Tyr Ser Gly Asn Al - #a Tyr Asp Leu Ser Gln 180 - # 185 - # 190 - - Val Gln Cys Leu Ala Tyr Ala Val Asn Ser Se - #r Asp Pro Leu Leu Ile 195 - # 200 - # 205 - - Glu Trp Lys Lys Tyr Glu Gly Asn Pro Val Le - #u Leu Pro Pro Pro Gly 210 - # 215 - # 220 - - Val Gly Tyr Lys Asp Phe Arg Asp Pro Ser Th - #r Leu Trp Ser Gly Pro 225 2 - #30 2 - #35 2 -#40 - - Asp Gly Glu Tyr Arg Met Val Met Gly Ser Ly - #s His Asn Glu ThrIle 245 - # 250 - # 255 - - Gly Cys Ala Leu Ile Tyr His Thr Thr Asn Ph - #e Thr His Phe Glu Leu 260 - # 265 - # 270 - - Lys Glu Glu Val Leu His Ala Val Pro His Th - #r Gly Met Trp Glu Cys 275 - # 280 - # 285 - - Val Asp Leu Tyr Pro Val Ser Thr Val His Th - #r Asn Gly Leu Asp Met 290 - # 295 - # 300 - - Val Asp Asn Gly Pro Asn Val Lys Tyr Val Le - #u Lys Gln Ser Gly Asp 305 3 - #10 3 - #15 3 -#20 - - Glu Asp Arg His Asp Trp Tyr Ala Ile Gly Se - #r Tyr Asp Ile ValAsn 325 - # 330 - # 335 - - Asp Lys Trp Tyr Pro Asp Asp Pro Glu Asn As - #p Val Gly Ile Gly Leu 340 - # 345 - # 350 - - Arg Tyr Asp Phe Gly Lys Phe Tyr Ala Ser Ly - #s Thr Phe Tyr Asp Gln 355 - # 360 - # 365 - - His Lys Lys Arg Arg Val Leu Trp Gly Tyr Va - #l Gly Glu Thr Asp Pro 370 - # 375 - # 380 - - Gln Lys Tyr Asp Leu Ser Lys Gly Trp Ala As - #n Ile Leu Asn Ile Pro 385 3 - #90 3 - #95 4 -#00 - - Arg Thr Val Val Leu Asp Leu Glu Thr Lys Th - #r Asn Leu Ile GlnTrp 405 - # 410 - # 415 - - Pro Ile Glu Glu Thr Glu Asn Leu Arg Ser Ly - #s Lys Tyr Asp Glu Phe 420 - # 425 - # 430 - - Lys Asp Val Glu Leu Arg Pro Gly Ala Leu Va - #l Pro Leu Glu Ile Gly 435 - # 440 - # 445 - - Thr Ala Thr Gln Leu Asp Ile Val Ala Thr Ph - #e Glu Ile Asp Gln Lys 450 - # 455 - # 460 - - Met Leu Glu Ser Thr Leu Glu Ala Asp Val Le - #u Phe Asn Cys Thr Thr 465 4 - #70 4 - #75 4 -#80 - - Ser Glu Gly Ser Val Ala Arg Ser Val Leu Gl - #y Pro Phe Gly ValVal 485 - # 490 - # 495 - - Val Leu Ala Asp Ala Gln Arg Ser Glu Gln Le - #u Pro Val Tyr Phe Tyr 500 - # 505 - # 510 - - Ile Ala Lys Asp Ile Asp Gly Thr Ser Arg Th - #r Tyr Phe Cys Ala Asp 515 - # 520 - # 525 - - Glu Thr Arg Ser Ser Lys Asp Val Ser Val Gl - #y Lys Trp Val Tyr Gly 530 - # 535 - # 540 - - Ser Ser Val Pro Val Leu Pro Gly Glu Lys Ty - #r Asn Met Arg Leu Leu 545 5 - #50 5 - #55 5 -#60 - - Val Asp His Ser Ile Val Glu Gly Phe Ala Gl - #n Asn Gly Arg ThrVal 565 - # 570 - # 575 - - Val Thr Ser Arg Val Tyr Pro Thr Lys Ala Il - #e Tyr Asn Ala Ala Lys 580 - # 585 - # 590 - - Val Phe Leu Phe Asn Asn Ala Thr Gly Ile Se - #r Val Lys Ala Ser Ile 595 - # 600 - # 605 - - Lys Ile Trp Lys Met Gly Glu Ala Glu Leu As - #n Pro Phe Pro Leu Pro 610 - # 615 - # 620 - - Gly Trp Thr Phe Glu Leu 625 6 - #30 - - - - (2) INFORMATION FOR SEQ ID NO: 3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2003 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia 1 (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pFFT111 - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:29..1873 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 3: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 3: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3: - - GGGACGAGTA CCAGTCCAGT CAGTCACC ATG CAA ACC CCT GAA - #CCC TTT ACA 52 - # Met Gln Thr - #Pro Glu Pro Phe Thr - # - # 635 - - GAC CTT GAA CAT GAA CCC CAC ACA CCC CTA CT - #G GAC CAC CAC CAC AAC 100 Asp Leu Glu His Glu Pro His Thr Pro Leu Le - #u Asp His His His Asn 640 - # 645 - # 650 - - CCA CCA CCA CAA ACC ACC ACA AAA CCT TTG TT - #C ACC AGG GTT GTG TCC 148 Pro Pro Pro Gln Thr Thr Thr Lys Pro Leu Ph - #e Thr Arg Val Val Ser 655 6 - #60 6 - #65 6 -#70 - - GGT GTC ACC TTT GTT TTA TTC TTC TTT GGT TT - #C GCT ATC GTA TTCATT 196 Gly Val Thr Phe Val Leu Phe Phe Phe Gly Ph - #e Ala Ile Val Phe Ile 675 - # 680 - # 685 - - GTT CTC AAC CAA CAG AAT TCT TCT GTT CGT AT - #C GTC ACC AAT TCG GAG 244 Val Leu Asn Gln Gln Asn Ser Ser Val Arg Il - #e Val Thr Asn Ser Glu 690 - # 695 - # 700 - - AAA TCT TTT ATA AGG TAT TCG CAG ACC GAT CG - #C TTG TCG TGG GAA CGG 292 Lys Ser Phe Ile Arg Tyr Ser Gln Thr Asp Ar - #g Leu Ser Trp Glu Arg 705 - # 710 - # 715 - - ACC GCT TTT CAT TTT CAG CCT GCC AAG AAT TT - #T ATT TAC GAT CCA GAT 340 Thr Ala Phe His Phe Gln Pro Ala Lys Asn Ph - #e Ile Tyr Asp Pro Asp 720 - # 725 - # 730 - - GGT CAG TTG TTT CAC ATG GGC TGG TAC CAT AT - #G TTC TAT CAA TAC AAC 388 Gly Gln Leu Phe His Met Gly Trp Tyr His Me - #t Phe Tyr Gln Tyr Asn 735 7 - #40 7 - #45 7 -#50 - - CCA TAC GCA CCG GTT TGG GGC AAT ATG TCA TG - #G GGT CAC TCA GTGTCC 436 Pro Tyr Ala Pro Val Trp Gly Asn Met Ser Tr - #p Gly His Ser Val Ser 755 - # 760 - # 765 - - AAA GAC ATG ATC AAC TGG TAC GAG CTG CCA GT - #C GCT ATG GTC CCG ACC 484 Lys Asp Met Ile Asn Trp Tyr Glu Leu Pro Va - #l Ala Met Val Pro Thr 770 - # 775 - # 780 - - GAA TGG TAT GAT ATC GAG GGC GTC TTA TCC GG - #G TCT ACC ACG GTC CTT 532 Glu Trp Tyr Asp Ile Glu Gly Val Leu Ser Gl - #y Ser Thr Thr Val Leu 785 - # 790 - # 795 - - CCA AAC GGT CAG ATC TTT GCA TTG TAT ACT GG - #G AAC GCT AAT GAT TTT 580 Pro Asn Gly Gln Ile Phe Ala Leu Tyr Thr Gl - #y Asn Ala Asn Asp Phe 800 - # 805 - # 810 - - TCC CAA TTA CAA TGC AAA GCT GTA CCC GTA AA - #C TTA TCT GAC CCG CTT 628 Ser Gln Leu Gln Cys Lys Ala Val Pro Val As - #n Leu Ser Asp Pro Leu 815 8 - #20 8 - #25 8 -#30 - - CTT ATT GAG TGG GTC AAG TAT GAG GAT AAC CC - #A ATC CTG TAC ACTCCA 676 Leu Ile Glu Trp Val Lys Tyr Glu Asp Asn Pr - #o Ile Leu Tyr Thr Pro 835 - # 840 - # 845 - - CCA GGG ATT GGG TTA AAG GAC TAT CGG GAC CC - #G TCA ACA GTC TGG ACA 724 Pro Gly Ile Gly Leu Lys Asp Tyr Arg Asp Pr - #o Ser Thr Val Trp Thr 850 - # 855 - # 860 - - GGT CCC GAT GGA AAG CAT AGG ATG ATC ATG GG - #A ACT AAA CGT GGC AAT 772 Gly Pro Asp Gly Lys His Arg Met Ile Met Gl - #y Thr Lys Arg Gly Asn 865 - # 870 - # 875 - - ACA GGC ATG GTA CTT GTT TAC TAT ACC ACT GA - #T TAC ACG AAC TAC GAG 820 Thr Gly Met Val Leu Val Tyr Tyr Thr Thr As - #p Tyr Thr Asn Tyr Glu 880 - # 885 - # 890 - - TTG TTG GAT GAG CCG TTG CAC TCT GTT CCC AA - #C ACC GAT ATG TGG GAA 868 Leu Leu Asp Glu Pro Leu His Ser Val Pro As - #n Thr Asp Met Trp Glu 895 9 - #00 9 - #05 9 -#10 - - TGC GTC GAC TTT TAC CCG GTT TCG TTA ACC AA - #T GAT AGT GCA CTTGAT 916 Cys Val Asp Phe Tyr Pro Val Ser Leu Thr As - #n Asp Ser Ala Leu Asp 915 - # 920 - # 925 - - ATG GCG GCC TAT GGG TCG GGT ATC AAA CAC GT - #T ATT AAA GAA AGT TGG 964 Met Ala Ala Tyr Gly Ser Gly Ile Lys His Va - #l Ile Lys Glu Ser Trp 930 - # 935 - # 940 - - GAG GGA CAT GGA ATG GAT TGG TAT TCA ATC GG - #G ACA TAT GAC GCG ATA 1012 Glu Gly His Gly Met Asp Trp Tyr Ser Ile Gl - #y Thr Tyr Asp Ala Ile 945 - # 950 - # 955 - - AAT GAT AAA TGG ACT CCC GAT AAC CCG GAA CT - #A GAT GTC GGT ATC GGG 1060 Asn Asp Lys Trp Thr Pro Asp Asn Pro Glu Le - #u Asp Val Gly Ile Gly 960 - # 965 - # 970 - - TTA CGG TGC GAT TAC GGG AGG TTT TTT GCA TC - #A AAG AGT CTT TAT GAC 1108 Leu Arg Cys Asp Tyr Gly Arg Phe Phe Ala Se - #r Lys Ser Leu Tyr Asp 975 9 - #80 9 - #85 9 -#90 - - CCA TTG AAG AAA AGG AGG ATC ACT TGG GGT TA - #T GTT GGA GAA TCAGAT 1156 Pro Leu Lys Lys Arg Arg Ile Thr Trp Gly Ty - #r Val Gly Glu Ser Asp 995 - # 1000 - # 1005 - - AGT GCT GAT CAG GAC CTC TCT AGA GGA TGG GC - #T ACT GTT TAT AAT GTT 1204 Ser Ala Asp Gln Asp Leu Ser Arg Gly Trp Al - #a Thr Val Tyr Asn Val 1010 - # 1015 - # 1020 - - GGA AGA ACA ATT GTA CTA GAT AGA AAG ACC GG - #G ACC CAT TTA CTT CAT 1252 Gly Arg Thr Ile Val Leu Asp Arg Lys Thr Gl - #y Thr His Leu Leu His 1025 - # 1030 - # 1035 - - TGG CCC GTT GAG GAA GTC GAG AGT TTG AGA TA - #C AAC GGT CAG GAG TTT 1300 Trp Pro Val Glu Glu Val Glu Ser Leu Arg Ty - #r Asn Gly Gln Glu Phe 1040 - # 1045 - # 1050 - - AAA GAG ATC AAG CTA GAG CCC GGT TCA ATC AT - #T CCA CTC GAC ATA GGC 1348 Lys Glu Ile Lys Leu Glu Pro Gly Ser Ile Il - #e Pro Leu Asp Ile Gly 1055 1060 - # 1065 - # 1070 - - ACG GCT ACA CAG TTG GAC ATA GTT GCA ACA TT - #T GAG GTG GAT CAA GCA 1396 Thr Ala Thr Gln Leu Asp Ile Val Ala Thr Ph - #e Glu Val Asp Gln Ala 1075 - # 1080 - # 1085 - - GCG TTG AAC GCG ACA AGT GAA ACC GAT GAT AT - #T TAT GGT TGC ACC ACT 1444 Ala Leu Asn Ala Thr Ser Glu Thr Asp Asp Il - #e Tyr Gly Cys Thr Thr 1090 - # 1095 - # 1100 - - AGC TTA GGT GCA GCC CAA AGG GGA AGT TTG GG - #A CCA TTT GGT CTT GCG 1492 Ser Leu Gly Ala Ala Gln Arg Gly Ser Leu Gl - #y Pro Phe Gly Leu Ala 1105 - # 1110 - # 1115 - - GTT CTA GCC GAT GGA ACC CTT TCT GAG TTA AC - #T CCG GTT TAT TTC TAT 1540 Val Leu Ala Asp Gly Thr Leu Ser Glu Leu Th - #r Pro Val Tyr Phe Tyr 1120 - # 1125 - # 1130 - - ATA GCT AAA AAG GCA GAT GGA GGT GTG TCG AC - #A CAT TTT TGT ACC GAT 1588 Ile Ala Lys Lys Ala Asp Gly Gly Val Ser Th - #r His Phe Cys Thr Asp 1135 1140 - # 1145 - # 1150 - - AAG CTA AGG TCA TCA CTA GAT TAT GAT GGG GA - #G AGA GTG GTG TAT GGG 1636 Lys Leu Arg Ser Ser Leu Asp Tyr Asp Gly Gl - #u Arg Val Val Tyr Gly 1155 - # 1160 - # 1165 - - GGC ACT GTT CCT GTG TTA GAT GAT GAA GAA CT - #C ACA ATG AGG CTA TTG 1684 Gly Thr Val Pro Val Leu Asp Asp Glu Glu Le - #u Thr Met Arg Leu Leu 1170 - # 1175 - # 1180 - - GTG GAT CAT TCG ATA GTG GAG GGG TTT GCG CA - #A GGA GGA AGG ACG GTT 1732 Val Asp His Ser Ile Val Glu Gly Phe Ala Gl - #n Gly Gly Arg Thr Val 1185 - # 1190 - # 1195 - - ATA ACA TCA AGG GCG TAT CCA ACA AAA GCG AT - #A TAC GAA CAA GCG AAG 1780 Ile Thr Ser Arg Ala Tyr Pro Thr Lys Ala Il - #e Tyr Glu Gln Ala Lys 1200 - # 1205 - # 1210 - - CTG TTC TTG TTC AAC AAC GCC ACA GGT ACG AG - #T GTG AAA GCA TCT CTC 1828 Leu Phe Leu Phe Asn Asn Ala Thr Gly Thr Se - #r Val Lys Ala Ser Leu 1215 1220 - # 1225 - # 1230 - - AAG ATT TGG CAA ATG GCT TCT GCA CCA ATT CA - #T CAA TAC CCT TTT 1873 Lys Ile Trp Gln Met Ala Ser Ala Pro Ile Hi - #s Gln Tyr Pro Phe 1235 - # 1240 - # 1245 - - TAATTACCGG CTATCGCTAT CCTTTTTGTT ATTGGTATTT ATGTATCTTA AT -#TTTCTTTT 1933 - - AAACCTTTTT ATTTGATAAA TATTAGTTCT TGTTATTGTG CTTCTAGTAA TA -#AATGAATG 1993 - - GTGTTATGGG - # - # - # 2003 - - - - (2) INFORMATION FOR SEQ ID NO: 4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 615 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4: - - Met Gln Thr Pro Glu Pro Phe Thr Asp Leu Gl - #u His Glu Pro His Thr 1 5 - # 10 - # 15 - - Pro Leu Leu Asp His His His Asn Pro Pro Pr - #o Gln Thr Thr Thr Lys 20 - # 25 - # 30 - - Pro Leu Phe Thr Arg Val Val Ser Gly Val Th - #r Phe Val Leu Phe Phe 35 - # 40 - # 45 - - Phe Gly Phe Ala Ile Val Phe Ile Val Leu As - #n Gln Gln Asn Ser Ser 50 - # 55 - # 60 - - Val Arg Ile Val Thr Asn Ser Glu Lys Ser Ph - #e Ile Arg Tyr Ser Gln 65 - # 70 - # 75 - # 80 - - Thr Asp Arg Leu Ser Trp Glu Arg Thr Ala Ph - #e His Phe Gln Pro Ala 85 - # 90 - # 95 - - Lys Asn Phe Ile Tyr Asp Pro Asp Gly Gln Le - #u Phe His Met Gly Trp 100 - # 105 - # 110 - - Tyr His Met Phe Tyr Gln Tyr Asn Pro Tyr Al - #a Pro Val Trp Gly Asn 115 - # 120 - # 125 - - Met Ser Trp Gly His Ser Val Ser Lys Asp Me - #t Ile Asn Trp Tyr Glu 130 - # 135 - # 140 - - Leu Pro Val Ala Met Val Pro Thr Glu Trp Ty - #r Asp Ile Glu Gly Val 145 1 - #50 1 - #55 1 -#60 - - Leu Ser Gly Ser Thr Thr Val Leu Pro Asn Gl - #y Gln Ile Phe AlaLeu 165 - # 170 - # 175 - - Tyr Thr Gly Asn Ala Asn Asp Phe Ser Gln Le - #u Gln Cys Lys Ala Val 180 - # 185 - # 190 - - Pro Val Asn Leu Ser Asp Pro Leu Leu Ile Gl - #u Trp Val Lys Tyr Glu 195 - # 200 - # 205 - - Asp Asn Pro Ile Leu Tyr Thr Pro Pro Gly Il - #e Gly Leu Lys Asp Tyr 210 - # 215 - # 220 - - Arg Asp Pro Ser Thr Val Trp Thr Gly Pro As - #p Gly Lys His Arg Met 225 2 - #30 2 - #35 2 -#40 - - Ile Met Gly Thr Lys Arg Gly Asn Thr Gly Me - #t Val Leu Val TyrTyr 245 - # 250 - # 255 - - Thr Thr Asp Tyr Thr Asn Tyr Glu Leu Leu As - #p Glu Pro Leu His Ser 260 - # 265 - # 270 - - Val Pro Asn Thr Asp Met Trp Glu Cys Val As - #p Phe Tyr Pro Val Ser 275 - # 280 - # 285 - - Leu Thr Asn Asp Ser Ala Leu Asp Met Ala Al - #a Tyr Gly Ser Gly Ile 290 - # 295 - # 300 - - Lys His Val Ile Lys Glu Ser Trp Glu Gly Hi - #s Gly Met Asp Trp Tyr 305 3 - #10 3 - #15 3 -#20 - - Ser Ile Gly Thr Tyr Asp Ala Ile Asn Asp Ly - #s Trp Thr Pro AspAsn 325 - # 330 - # 335 - - Pro Glu Leu Asp Val Gly Ile Gly Leu Arg Cy - #s Asp Tyr Gly Arg Phe 340 - # 345 - # 350 - - Phe Ala Ser Lys Ser Leu Tyr Asp Pro Leu Ly - #s Lys Arg Arg Ile Thr 355 - # 360 - # 365 - - Trp Gly Tyr Val Gly Glu Ser Asp Ser Ala As - #p Gln Asp Leu Ser Arg 370 - # 375 - # 380 - - Gly Trp Ala Thr Val Tyr Asn Val Gly Arg Th - #r Ile Val Leu Asp Arg 385 3 - #90 3 - #95 4 -#00 - - Lys Thr Gly Thr His Leu Leu His Trp Pro Va - #l Glu Glu Val GluSer 405 - # 410 - # 415 - - Leu Arg Tyr Asn Gly Gln Glu Phe Lys Glu Il - #e Lys Leu Glu Pro Gly 420 - # 425 - # 430 - - Ser Ile Ile Pro Leu Asp Ile Gly Thr Ala Th - #r Gln Leu Asp Ile Val 435 - # 440 - # 445 - - Ala Thr Phe Glu Val Asp Gln Ala Ala Leu As - #n Ala Thr Ser Glu Thr 450 - # 455 - # 460 - - Asp Asp Ile Tyr Gly Cys Thr Thr Ser Leu Gl - #y Ala Ala Gln Arg Gly 465 4 - #70 4 - #75 4 -#80 - - Ser Leu Gly Pro Phe Gly Leu Ala Val Leu Al - #a Asp Gly Thr LeuSer 485 - # 490 - # 495 - - Glu Leu Thr Pro Val Tyr Phe Tyr Ile Ala Ly - #s Lys Ala Asp Gly Gly 500 - # 505 - # 510 - - Val Ser Thr His Phe Cys Thr Asp Lys Leu Ar - #g Ser Ser Leu Asp Tyr 515 - # 520 - # 525 - - Asp Gly Glu Arg Val Val Tyr Gly Gly Thr Va - #l Pro Val Leu Asp Asp 530 - # 535 - # 540 - - Glu Glu Leu Thr Met Arg Leu Leu Val Asp Hi - #s Ser Ile Val Glu Gly 545 5 - #50 5 - #55 5 -#60 - - Phe Ala Gln Gly Gly Arg Thr Val Ile Thr Se - #r Arg Ala Tyr ProThr 565 - # 570 - # 575 - - Lys Ala Ile Tyr Glu Gln Ala Lys Leu Phe Le - #u Phe Asn Asn Ala Thr 580 - # 585 - # 590 - - Gly Thr Ser Val Lys Ala Ser Leu Lys Ile Tr - #p Gln Met Ala Ser Ala 595 - # 600 - # 605 - - Pro Ile His Gln Tyr Pro Phe 610 - # 615 - - - - (2) INFORMATION FOR SEQ ID NO: 5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pSST103 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 5: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 5: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5: - - GARCARYTNC CNGTNTAYTT YTAYATHGCN AAR - # - # 33 - - - - (2) INFORMATION FOR SEQ ID NO: 6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pFFT111 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 6: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 6: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6: - - GGNTGGGCNA CNGTNTAYAA Y - # - # - #21 - - - - (2) INFORMATION FOR SEQ ID NO: 7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pFFT111 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 7: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 7: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7: - - ATHGTNGARG GNTTYGCNCA R - # - # - #21 - - - - (2) INFORMATION FOR SEQ ID NO: 8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pFFT111 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 8: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 8: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8: - - CCGAATTCAA TACGACTCAC TATAGCG - # - # 27 - - - - (2) INFORMATION FOR SEQ ID NO: 9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pSST103 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 9: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 9: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9: - - Ala Asp Val Leu Phe Xaa Xaa Thr Thr Ser Gl - #u Gly Ser Val Ala Arg 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID NO: 10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pSST103 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 10: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 10: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10: - - Glu Gln Leu Pro Val Tyr Phe Tyr Ile Ala Ly - #s 1 5 - # 10 - - - - (2) INFORMATION FOR SEQ ID NO: 11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pFFT111 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 11: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 11: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11: - - Val Val Leu Asp Leu Glu Thr Lys 1 5 - - - - (2) INFORMATION FOR SEQ ID NO: 12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pSST103 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 12: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 12: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12: - - Phe Arg Asp Pro Ser Thr Leu Trp Leu Xaa Pr - #o Asp Gly Glu Tyr 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID NO: 13: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pSST103 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 13: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 13: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13: - - Gly Trp Ala Asn Ile Leu 1 5 - - - - (2) INFORMATION FOR SEQ ID NO: 14: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pFFT111 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 14: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 14: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14: - - Gly Trp Ala Thr Val Tyr Asn Val Gly Arg 1 5 - # 10 - - - - (2) INFORMATION FOR SEQ ID NO: 15: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pFFT111 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 15: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 15: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15: - - Leu Leu Val Asp His Ser Ile Val Glu Gly Ph - #e Ala Gln Gly Gly Arg 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID NO: 16: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Helianthus - #tuberosus (B) STRAIN: Columbia (D) DEVELOPMENTAL STAGE: - #Adult (F) TISSUE TYPE: Tuber (G) CELL TYPE: storage - #parenchyma - - (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda Uni - #-Zap XR (Stratagene, USA) (B) CLONE: pFFT111 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 89/12386 A1 (I) FILING DATE: 21-JUN - #-1989 (J) PUBLICATION DATE: 2 - #8-DEC-1989 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 16: FROM 1 TO 4100 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/04692 A1 (I) FILING DATE: 09-AUG - #-1993 (J) PUBLICATION DATE: 0 - #3-MAR-1994 (K) RELEVANT RESIDUES I - #N SEQ ID NO: 16: FROM 1 TO 1438 - - (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: WO - # 94/14970 A1 (I) FILING DATE: 28-DEC - #-1993 (J) PUBLICATION DATE: 0 - #7-JUL-1994 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16: - - Val Gly Glu Ser Asp Ser 1 5__________________________________________________________________________

Number	Date	Country	Kind
95200015	Jan 1995	EPX
95200762	Mar 1995	EPX

Number	Date	Country
8912386	Dec 1989	WOX
9414970	Jul 1994	WOX
9601904	Jan 1996	WOX

DNA sequences encoding carbohydrate polymer synthesizing enzymes and method for producing transgenic plants

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Entry
Stratagene Instruction Manual: UniZAP.TM. Library, Jan. 19, 1994, pp. 1-10.
Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Lab. Press, pp. 8.46-8.47, 1989.
Hellwege et al. Plant Journal 12(5):1057-1065, 1997.
Bournay et al. Nucl. Acids Res. 24(12):2347-2351, 1996.
Kossmann et al. Progress in Biotechnol. 10:271-278, 1995.
Angenent et al. "Purification and Properties of Sucrose Fructosyltransferases from Barley Leaves and Onion Seeds" by Angenent, G.C., et al., Insulin and Insulin-containing Crops, 1993, pp. 173-184, Fuchs, A., ed.
Praznik, W., et al., "Isolation and Characterization of Sucrose:Sucrose 1.sup.F -.beta.-D- Fructosyltransferase from Tubers of Helianthus tuberosus L." Agricultural Biol. Chem., vol. 54, No. 9, 1990, pp. 2429-2431.
Luscher, M., et al., "Purification and characterization of fructan: fructan fructosyltransferase from Jerusalem artichoke (Helianthus tuberosus L.)", New Phytol, vol. 123, 1993, pp. 717-724.
Koops, A., et al., "Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus "Colombia" I. Fructan:fructan fructosyltransferase" Journal of Experimental Botany, vol. 45, No. 280, Nov. 1994, pp. 1623-1631.