Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 184.108.40.206). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT–PCR, followed by rapid amplification of cDNA ends (RACE–PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.
Raffinose oligosaccharides (i.e. raffinose, stachyose and higher homologues; Fig. 1) represent important non-structural carbohydrates in plants. In Cucurbitaceae, Lamiaceae and other plant families, they serve as transport carbohydrates in the phloem, as storage reserves in leaves, roots and tubers, and possibly act as cryoprotectants in over-wintering species ( Keller & Pharr 1996). In the majority of plant species, however, synthesis of stachyose is restricted to seeds ( Dey 1985). While the seeds of monocotyledonous species contain raffinose as the major oligosaccharide, dicotyledons preferentially accumulate stachyose and higher homologues ( Kuo et al. 1988 ). Accumulation of raffinose oligosaccharides during seed development has been related to the acquisition of desiccation tolerance, and they may also provide an important source of carbon during germination ( Obendorf 1997). Their α-galactosidic linkages are indigestible by humans and thus raffinose oligosaccharides may contribute to flatulence ( Cristofaro et al. 1974 ). Although there is some debate about the potential health benefits of non-digestible oligosaccharides ( Delzenne & Roberfroid 1994), breeding programmes have been designed to reduce the levels of these oligosaccharides in seeds and their elimination from seed-derived products has been extensively studied ( Mansour & Khalil 1998; Slominski 1994).
The biosynthesis of raffinose oligosaccharides proceeds by stepwise transfer of galactose residues from an unusual donor, galactinol (O-α- d-galactopyranosyl-(1→1)- l-myo-inositol), to sucrose. Raffinose synthase (EC 220.127.116.11) transfers a galactose moiety from galactinol to sucrose, yielding raffinose and myo-inositol ( Lehle & Tanner 1973; Ohsumi et al. 1998 ). Stachyose synthase (EC 18.104.22.168) reversibly converts raffinose and galactinol to stachyose and myo-inositol, respectively ( Hoch et al. 1999 ; Holthaus & Schmitz 1991; Peterbauer & Richter 1998). Galactinol is recycled by the enzyme galactinol synthase (EC 22.214.171.124) through galactosyl transfer from UDP-galactose to myo-inositol ( Liu et al. 1995 ; Liu et al. 1998 ).
In seeds of many legumes, such as soybean (Glycine max), lentil (Lens culinaris) and chickpea (Cicer arietinum), galactosyl derivatives of d-ononitol (1 d-4-O-methyl-myo-inositol), d-pinitol (1 d-3-O-methyl-chiro-inositol) and/or d-chiro-inositol are present in addition to galactinol ( Horbowicz & Obendorf 1994; Obendorf 1997). These galactosyl cyclitols arise by galactosyl transfer from galactinol rather than from UDP-galactose ( Peterbauer et al. 1998 ). We recently demonstrated that there is a biochemical link between the metabolism of a range of galactosyl cyclitols and raffinose oligosaccharides ( Hoch et al. 1999 ; Peterbauer & Richter 1998). Stachyose synthase purified from adzuki bean (Vigna angularis) catalysed the galactosyl transfer from galactinol to d-ononitol, yielding galactosyl ononitol (O-α- d-galactopyranosyl-(1→3)-4-O-methyl- d-myo-inositol) ( Richter et al. 1997 ) and the co-product myo-inositol ( Fig. 1). The catalytic efficiency for galactosylation of ononitol was almost 1.9-fold higher compared to that of raffinose, providing evidence that this pathway is active in vivo ( Peterbauer & Richter 1998). Furthermore, the enzyme utilized the resulting galactosyl ononitol as a galactosyl donor to form stachyose from raffinose.
Stachyose synthase from lentil seeds was also found to be a multi-functional enzyme. It exhibited an even broader substrate specificity, utilizing d-pinitol (1 d-3-O-methyl-chiro-inositol), galactopinitol A (O-α- d-galactopyranosyl-(1→2)-4-O-methyl- d-chiro-inositol), d-chiro-inositol and d-ononitol in addition to galactinol and raffinose ( Hoch et al. 1999 ). Collectively, these results suggest that the accumulation of stachyose in seeds may involve multiple intermediates which are produced and also consumed, at least to a certain extent, by stachyose synthase.
Here we report on the cloning and functional expression of a cDNA encoding stachyose synthase from adzuki bean seeds. To our knowledge, this constitutes the first report of a nucleotide sequence encoding a stachyose synthase. In addition, polyclonal antibodies were raised against purified stachyose synthase and used to examine protein levels, together with transcript abundance, in developing and germinating seeds.
Cloning and analysis of the cDNA encoding stachyose synthase from adzuki bean
Stachyose synthase was purified from mature seeds of adzuki bean using a purification scheme reported previously ( Peterbauer & Richter 1998) and digested with lysine-C protease to obtain internal peptides for micro-sequencing. Ten internal peptide sequences were obtained and used to design degenerate primers to amplify partial cDNA fragments by RT–PCR. Total RNA from developing adzuki bean seeds, harvested about 20–23 days after flowering, was used as a template. Using several combinations of primers, three PCR fragments of about 0.2, 1.2 and 1.6 kb were amplified, cloned into the plasmid pCR2.1 and sequenced. The longest fragment was obtained by using the primers 5′-GCICCI(A/C)GITT(C/T)GTIGTIAT(T/C/A)GA(TC)G-3′ and 5′-TC(T/C)TG(A/G/T)ATIGTICCICCI(C/G) (T/A)(A/G)TT(G/A)AAC-3′, which were originally derived from the peptides APRFVVID and MFNSGGTIQE, respectively. The fragment encoded all known internal peptide sequences of the purified protein and contained the sequences of the other PCR fragments ( Fig. 2). The complete cDNA sequence was subsequently obtained by RACE–PCR ( Frohman et al. 1988 ).
The composed sequence of 3046 nucleotides (excluding a poly(A + ) tail of 28 nucleotides) was found to contain an open reading frame from nucleotide 343 to 2913. The deduced amino acid sequence corresponded to a polypeptide of 857 amino acid residues with a calculated molecular mass of 94.9 kDa and a pI of 5.39. The 5′ non-coding region contained three ATG codons upstream of the putative initiation codon, forming small open reading frames of 34 (nucleotides 20–124), 5 (nucleotides 126–143) and 19 amino acids (nucleotides 130–189), respectively. No polyadenylation signal identical with the animal consensus sequence AATAAA was found. A putative polyadenylation signal AATAAT is located 21 bp upstream from the poly(A + ) tail.
The N-terminus of the purified protein previously reported (NDPVNATLGLEPSEKVFDLLDGKL) ( Peterbauer & Richter 1998) was located at positions 5–28 of the deduced amino acid sequence, although Ser was found at position 24 instead of a Leu residue. Since all other peptide sequences obtained by digestion of stachyose synthase were represented within the predicted amino acid sequence, we conclude that the N-terminal peptide was also derived from the same gene and not from a related one. We found no evidence for a signal peptide, but five potential N-glycosylation sites Asn-X-Ser/Thr (where X represents any residue except proline) were detected. The gene encoding this cDNA was designated VaSTS1, for Vigna angularis stachyose synthase 1.
Comparison of the deduced amino acid sequence of VaSTS1 with sequences in the GenBank and EMBL databases revealed that it was related to a putative raffinose synthase or seed imbibition protein from Arabidopsis thaliana (accession no. AC007138) (53% identity) and to a raffinose synthase from Cucumis sativus (accession no. AF073744). Likewise, homologies were found between the deduced amino acid sequence of VaSTS1 and raffinose synthase from Glycine max and Vicia faba ( Watanabe & Oeda 1998). Overall homologies extended over the entire length of proteins, with single amino acid residues as well as several blocks of amino acids ( Fig. 2). The deduced amino acid sequence of VaSTS1 contained a central insertion not present in the other sequences. Significant scores were also obtained for a seed imbibition protein ( SIP1) from Hordeum vulgare (accession no. M77475), and several other putative seed imbibition proteins from Arabidopsis thaliana (accession nos Z26468, AC002328, AF007269and Z26507), Brassica oleracea (accession no. X79330) and Cicer arietinum (accession no. X95875), respectively.
Genomic DNA gel blot analysis was performed with a 1.1 kbp cDNA probe (nucleotides 347–1484) at high- stringency conditions ( Fig. 3a). Digestion with EcoRI, XbaI, and BglII produced two strong and one or two weak bands. Because none of these enzymes cut within the probe, these results suggest the presence of two highly homologous genes and probably one or two more distantly related genes. In support of this, EcoRI-digested DNA showed two additional strong bands when the blot was re-probed with a 2.0 kb cDNA fragment (nucleotides 347–2303) covering an EcoRI restriction site ( Fig. 3b).
Heterologous expression in insect cells
The entire coding region of VaSTS1 was amplified by PCR and subcloned into the pVL1393 baculovirus expression vector. The identity of the insert was verified by sequencing. The construct was introduced into Sf21 insect cells for expression under the control of the polyhedrin promotor. Lysates of cells infected with recombinant baculoviruses were harvested after four days and analysed by Western blotting ( Fig. 4). Polyclonal antibodies raised against stachyose synthase isolated from adzuki bean seeds cross-reacted with a polypeptide with an apparent molecular mass of about 98 kDa, which migrated to exactly the same position as the purified protein from adzuki bean. This protein was not present in Sf21 insect cells grown under identical conditions.
The catalytic properties of the recombinant protein were investigated by incubation of cell lysates with various substrates ( Fig. 5). Reaction products were analysed by high-performance liquid chromatography with pulsed amperometric detection (HPLC–PAD) and their identity was confirmed by gas chromatography/mass spectroscopy (GC–MS) of respective trimethylsilyl derivatives. When assayed with galactinol and raffinose, stachyose and myo-inositol were formed at a rate of 0.80 nkat mg− 1 total protein. d-Ononitol could replace raffinose to give galactosyl ononitol and the co-product myo-inositol (at a rate of 0.75 nkat mg− 1). When assayed with galactosyl ononitol and raffinose, stachyose and d-ononitol were formed (0.33 nkat mg− 1). Lysates of uninfected insect cells grown under identical conditions were devoid of any of these enzyme activities (data not shown).
Changes in transcript, protein and carbohydrate levels during seed development and germination
Transcript levels were studied in seeds harvested at representative growth stages during development and in germinating seeds. Figure 6(a) shows that transcripts are only abundant during a fairly short period about midway through seed development. A hybridization signal of about 3 kbp appeared at growth stage III (17–19 days after flowering), reached its highest levels at growth stage IV (22–22 days after flowering), and declined thereafter. When more sensitive RT–PCR was used, low levels of transcripts were detected in seeds harvested at maturity, but not in seeds stored for prolonged periods of time (data not shown). Western analysis ( Fig. 6b) and activity assays ( Fig. 6c) showed that the accumulation of the enzyme commenced in parallel with transcript levels, but protein and activity levels continued to increase during maturation. Likewise, galactosyl ononitol and stachyose were initially detected at growth stage III, together with galactinol and raffinose ( Fig. 6d). Stachyose increased to 50.1 μmol g− 1 fresh mass in dormant seeds. Galactosyl ononitol was only moderately accumulated, reaching 8.2 μmol g− 1 fresh mass, while galactinol and raffinose levels remained low.
By using RNA blot as well as RT–PCR techniques, we were unable to detect transcripts in total RNA prepared from germinating seeds (data not shown). However, appreciable levels of stachyose synthase were maintained during the first stages of germination ( Fig. 7a). Stachyose synthase activity declined from 4.5 nkat g− 1 fresh mass in dry seeds to 0.6 nkat g− 1 fresh mass in cotyledons harvested 7 days after imbibition ( Fig. 7b). Neither a cross-reactive protein nor stachyose synthase activity were detected in roots and shoots (data not shown). Stachyose increased slightly during the first hours of imbibition and declined to undetectable levels at day 7 after imbibition ( Fig. 7c). The levels of galactosyl ononitol, galactinol and raffinose were almost constant during the first day after imbibiton and declined thereafter.
In this study, we have cloned a cDNA encoding for stachyose synthase from adzuki bean seeds. The identity of the cDNA was verified by functional expression in insect cells using the baculovirus/insect cell system. Insect cells synthesize neither stachyose nor its precursors and are therefore an ideal eukaryotic system for heterologous expression of stachyose synthase. Western blotting and activity assays provided compelling evidence that the cDNA encodes a functional stachyose synthase. The recombinant enzyme catalysed the synthesis of galactosyl ononitol from galactinol and d-ononitol and was able to utilize galactosyl ononitol as a donor to form stachyose from raffinose. The rates of the latter reactions (92.7 and 40.8% compared with the synthesis of stachyose from galactinol and raffinose) were almost identical to those of native stachyose synthase from adzuki bean (103.5 and 37.8%, respectively) ( Peterbauer & Richter 1998). On 5% SDS gels, the apparent molecular mass of both the recombinant and native enzyme was about 98 kDa, which is in fairly good agreement with the molecular mass of 94.9 kDa deduced from the amino acid sequence of the cDNA. However, using gradient gels, the apparent molecular mass of stachyose synthase purified from adzuki bean was only about 90 kDa ( Peterbauer & Richter 1998). The reason for the aberrant migration pattern of stachyose synthase is not yet clear.
The amino acid sequence of the enzyme shares homology with raffinose synthase, an enzyme catalysing a very similar reaction. Both stachyose synthase and raffinose synthase recognize galactinol as galactosyl donor and utilize acceptors that differ only in one galactose unit. However, despite these molecular and biochemical similarities, stachyose synthase is totally inactive on sucrose ( Hoch et al. 1999 ; Peterbauer & Richter 1998), while raffinose synthase is inactive on raffinose as acceptor ( Lehle & Tanner 1973). It will be of interest to identify regions responsible for the specific recognition of galactose acceptors.
Fructosyl transferases, which catalyse the synthesis of fructans from sucrose, are related to invertases and are thought to have evolved from the latter by small mutational changes ( Hellwege et al. 1997 ; Sprenger et al. 1995 ; van der Meer et al. 1998 ). In contrast, no homologies between the amino acid sequence of stachyose synthase and those of α-galactosidases were found. However, to date, only acidic α-galactosidases with a broad substrate specificity towards oligo-and polysaccharides have been cloned ( Davis et al. 1996 ; Davis et al. 1996 ; Overbeeke et al. 1989 ; Zhu & Goldstein 1994). Distinct α-galactosidases with high specificity towards raffinose oligosaccharides and alkaline pH optima have been identified in leaves of Cucurbitaceae ( Gao & Schaffer 1999; Gaudreault & Webb 1983), but the amino acid sequences of the latter α-galactosidases have not yet been reported. Stachyose synthase and raffinose synthase are related to a family of seed imbibition proteins of unknown function. It will be interesting to see whether these proteins are glycosyl transferases or hydrolases.
Stachyose synthase gene expression as well as protein and activity levels were studied during development and germination of adzuki bean. Transcripts were initially detected during mid- to late stages of development, in parallel with stachyose synthase protein and activity. Their appearance coincided with that of galactinol and raffinose (the substrates of the enzyme), as well as with that of stachyose and galactosyl ononitol (its products). In summary, these data suggest that the enzymes of stachyose synthesis (i.e. galactinol synthase, raffinose synthase and stachyose synthase) were coordinately expressed during seed development. However, no VaSTS1 mRNA was detected in stored and germinating seeds, although fairly high levels of stachyose synthase protein and activity were found. These results suggest that the protein synthesized during development remained stable in resting seeds and was slowly degraded during germination. However, RNA blot analysis may not detect low levels of VaSTS1 expression, while more sensitive RT–PCR may fail to detect expression of homologous gene(s), which could also contribute to the synthesis of the protein.
The levels of stachyose present in the seeds were fairly well mirrored by stachyose synthase levels during seed development. However, comparison of the pattern of stachyose and galactosyl ononitol accumulation clearly demonstrated that the synthesis of stachyose followed complex kinetics in situ. Galactosyl ononitol initially increased faster than that of stachyose, but remained considerably lower during later stages of maturation, although both substances are synthesized by one enzyme. A similar pattern of galactosyl ononitol and stachyose accumulation has been reported for rice bean ( Peterbauer et al. 1998 ). Initial stachyose synthesis might have been limited by raffinose availability during early stages of raffinose oligosaccharide accumulation, while galactosyl ononitol synthesis was much less likely to be restricted by substrate supply, since high concentrations of d-ononitol are already present in young seeds ( Peterbauer et al. 1998 ). On the other hand, levels of galactosyl ononitol are not only determined by the rate of synthesis from d-ononitol, but also by the rate of galactosyl transfer from galactosyl ononitol to raffinose.
It is also important to note that stachyose synthase activity several-fold exceeded the level needed to explain the content of products present in mature seeds. The enzymatic capacity of mature adzuki bean seeds was sufficient to synthesize the amount of stachyose present in about 3 h, and to synthesize the actual amount of galactosyl ononitol within less than 1 h. To reconcile activities with product levels, one has to consider that all reactions catalysed by stachyose synthase are reversible at physiological substrate concentrations ( Peterbauer et al. 1998 ; Tanner & Kandler 1968). The mass action ratio for stachyose synthesis from galactinol and raffinose increased from 2.1 at growth stage III to about 4.7 in mature seeds (data not shown). The latter value corresponds to the thermodynamic equilibrium of the reaction, which is between 4.0 and 5.0 ( Tanner & Kandler 1968). These results suggest that excess stachyose synthase activity was needed to compensate for reverse reactions. However, flux through an equilibrium reaction is dependent on actual metabolite concentrations ( Stitt 1999). Thus, the rate of stachyose accumulation may not only be controlled by stachyose synthase, but also by the levels of galactinol, myo-inositol, raffinose, and, in the case of adzuki bean, d-ononitol.
It is currently unclear whether the presence of stachyose synthase activity in germinating seeds is of physiological significance. Raffinose oligosaccharides and galactosyl cyclitols are believed to be degraded by α-galactosidases during germination ( Keller & Pharr 1996). High α-galactosidase activity is present in germinating seeds and hydrolysis of raffinose oligosaccharides is irreversible under physiological conditions ( Dhar et al. 1994 ; Porter et al. 1990 ). However, the removal of the terminal galactosyl residue is the rate-limiting step in the hydrolysis of stachyose to sucrose and galactose ( Porter et al. 1990 ). Since conversion of one molecule of stachyose to raffinose and galactinol (or galactosyl ononitol) doubles the amount of substrates for α-galactosidases, stachyose synthase operating in the reverse direction may accelerate the rate of stachyose breakdown in germinating seeds.
In summary, our results suggest that stachyose levels in developing and germinating seeds might by regulated by complex interactions between anabolic and catabolic enzymes and metabolite levels. The data presented in this work represent the first molecular description of a stachyose synthase gene and will allow cloning of genes encoding stachyose synthase and related galactosyl transferases from other species and plant organs, and investigation of the regulation of these enzymes on a molecular level.
Seeds of adzuki bean (Vigna angularis Ohwi et Ohashi) were obtained from a local market. Plants were grown in commercial soil in a growth chamber with 14 h of light at 24°C (70% relative humidity) and 10 h dark at 18°C (80% relative humidity). To promote flowering, the light period was reduced to 11 h after 4 weeks. Developing seeds were collected at different times thereafter. For germination, seeds were imbibed in water for 6 h and placed on wet filter paper in Petri dishes. Samples were used immediately or stored at − 70°C after freezing in liquid N2. Galactinol, d-ononitol and galactosyl ononitol were isolated as described in previous studies ( Peterbauer et al. 1998 ; Richter et al. 1997 ).
Determination of partial amino acid sequences and production of polyclonal antibodies
Stachyose synthase was purified from mature seeds of adzuki bean as previously described ( Peterbauer & Richter 1998). For micro-sequencing, a sample of the protein was submitted to the Institute of Biochemistry (Medicinal Faculty), University of Vienna. The protein was separated on a PAGE gel ( Laemmli 1970) and electroblotted to a polyvinylidene difluoride (PVDF) membrane. The protein band was cut out and digested with lysine-C protease. Peptides were purified by reversed-phase HPLC and sequenced by automated Edman degradation. One milligram of the purified protein was used to raise polyclonal antibodies in rabbits (Biogenes Inc., Berlin, Germany). The IgG fraction from the sera was purified by affinity chromatography.
Enzyme, protein and carbohydrate assay
Total soluble plant protein was extracted with 100 m m Na-phosphate (pH 7.0), 1 m m dithiothreitol (DTT) and protease inhibitors (Complete protease inhibitor cocktail, Boehringer Mannheim) as described previously ( Peterbauer et al. 1998 ). Extracts were desalted into 50 m m Na-phosphate (pH 7.0), 1 m m DTT, using the method of Helmerhorst & Stokes (1980). Enzyme assays were carried out at 30°C in reaction mixtures containing 50 m m Na-phosphate (pH 7.0), 1 m m DTT, 10 m m galactinol and 50 m m raffinose. Where specified, galactinol was replaced by galactosyl ononitol (10 m m) and raffinose was replaced by d-ononitol (20 m m). At intervals, aliquots were removed, heated to 100°C for 5 min, diluted 20-fold and analysed by HPLC–PAD on a Carbopac PA10 column (Dionex) using a Dionex DX500 chromatography system. To separate raffinose and stachyose, the column was thermostated at 30°C and eluted with 100 m m NaOH at a flow rate of 1 ml min− 1. To separate galactinol, myo-inositol, galactosyl ononitol and d-ononitol, the column was thermostated at 15°C and eluted with 20 m m NaOH at a flow rate of 0.75 ml min− 1 ( Peterbauer et al. 1998 ). To confirm the identity of reaction products, aliquots of assays were evaporated to dryness, converted into trimethylsilyl derivatives, and analysed by GC–MS ( Peterbauer & Richter 1998). Total protein concentration was determined by the method of Bradford (1976), with bovine serum albumin (BSA) as the standard. Soluble carbohydrates were assayed as described previously ( Peterbauer et al. 1998 ).
SDS–PAGE and Western blotting
Protein samples were subjected to SDS–PAGE on 5.0 or 7.5% polyacrylamide gels prepared according to Laemmli (1970) and blotted onto immun-blot PVDF membranes (BioRad) using a semi-dry system (NovaBlot, Pharmacia). The membranes were blocked over night at 4°C with 3% (w/v) BSA in 0.1 m Tris–HCl (pH 7.5), 0.9% (w/v) NaCl (TBS). After washing with TBS containing 0.05% (v/v) Tween-20, the membranes were incubated with the primary antibodies diluted 1:10 000 in TBS containing 1% (w/v) BSA. The membranes were washed twice with TBS containing 0.05% (v/v) Tween-20 and were then immersed in a solution of goat anti-rabbit IgG-alkaline phosphatase conjugate (Calbiochem) diluted 1:3000 in TBS containing 1% (w/v) BSA. The secondary antibody conjugate was visualized with a chromogenic reaction using nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) (Calbiochem).
RT–PCR, RACE–PCR and cloning of PCR products
Degenerate oligonucleotide primers were designed based on the partial amino acid sequence data. Except for three bases at the 3′ end, deoxyinosine was included at all positions of fourfold degeneracy. Total RNA was isolated from developing adzuki bean seeds by a modified proteinase K procedure ( Speirs & Longhurst 1993). First-strand cDNA synthesis was performed by incubating total RNA for 1 h at 48°C with AMV reverse transcriptase and an oligo-(dT) primer, using a reverse transcription system (Promega). PCR was performed on 10 μl of the first-strand synthesis reactions in total volumes of 50 μl, containing 10 m m Tris–HCl (pH 9.0), 50 m m KCl, 2 m m MgCl2, 0.1% Triton X-100, 0.2 m m dNTPs (Promega), 2 μm of each primer and 2 U Dynazyme II DNA polymerase (Finnzymes). After an initial denaturation at 94°C for 2.0 min, reaction mixtures were subjected to 40 cycles at 94°C for 1.0 min, 49°C for 1.5 min and 72°C for 2.0 min, followed by a final 8 min incubation at 72°C. PCR products were purified from agarose gels with a Qiaex II gel extraction kit (Qiagen), ligated into the linearized plasmid vector pCR2.1 (TA cloning kit, Invitrogen), and sequenced.
The cDNA ends were amplified by using RACE–PCR kits (Life Technologies) according to protocols provided by the manufacturer. For 3′-RACE, RNA was reverse-transcribed with an oligo-(dT) adaptor primer and used as template for PCR in conjunction with a specific primer (5′-GAGTATGTTGTGTACCTCAAT-3′) and a primer complementary to the adaptor primer. For 5′-RACE, RNA was reverse-transcribed with a specific antisense primer (5′-TCCTTCATCCCACCACACT-3′). An oligo-(dA) anchor sequence was added to the 3′ end of the reaction products. The 3′-RACE adaptor primer was installed by second-strand synthesis and PCR was performed using a specific antisense primer (5′-GCTTTGCAACAACACCCTCCACT-3′) and the primer complementary to the adaptor primer. PCR products were cloned as described above.
DNA sequencing and analysis
DNA sequences were determined by the dideoxy-chain termination method using the Prism Ready Reaction Dyedeoxy Terminator Cycle Sequencing kit (Perkin Elmer), a Hybaid OmnE thermocycler and an ABI Prism 310 sequencer (Perkin Elmer). Vector-derived or gene-specific oligonucleotides were used as primers. Analysis of DNA sequences was carried out using dnastar software. The blast program ( Altschul et al. 1997 ) was used to search the GenBank/EMBL sequence databases for homologous sequences.
Nucleic acid hybridization analysis
For genomic DNA blot analysis, 20 μg genomic DNA was digested with various restriction enzymes, separated in a 0.8% agarose gel, and blotted onto positively charged nylon membranes (Boehringer Mannheim). The blot was probed with VaSTS1 cDNA fragments that had been labelled by PCR using digoxigenin-11-dUTP (Boehringer Mannheim). Hybridization was carried out overnight at 42°C in DIG Easy hyb buffer (Boehringer Mannheim). Subsequently, the membrane was washed twice in 2 × SSC, 0.1% SDS at room temperature for 10 min and twice in 0.1 × SSC, 0.1% SDS at 68°C for 15 min. Bound probe was visualized with an anti-digoxigenin–alkaline phosphatase conjugate and chemoluminescence detection. HindIII-digested λ-DNA fragments were used as markers.
For RNA gel blot analysis, equal amounts of total RNA (10 μg) were size-fractionated on 1.2% agarose gels containing formaldehyde, and blotted. Hybridization was carried out as described above, except that the filter was hybridized at 50°C. The membranes were washed twice in 2 × SSC, 0.1% SDS at room temperature and twice in 0.5 × SSC, 0.1% SDS at 68°C. Transcript sizes were estimated with reference to digoxigenin-labelled RNA molecular size markers (Boehringer Mannheim).
Heterologous expression of recombinant VaSTS1 in insect cells
To engineer the entire coding region of VaSTS1 into the baculovirus transfer vector pVL1393 (PharMingen), PCR was performed on reverse-transcribed RNA using the Expand High Fidelity PCR system (Boehringer Mannheim). Gene-specific oligonucleotides were designed to introduce an XbaI restriction site at the 5′ end (5′-GCCGTCTAGACCATGGCTCCTCCG-3′) upstream of the ATG and a BglII restriction site (5′-GCC- AGATCTCATTGAAGCCTATGAA-3′) following the stop codon of the coding region. The conditions for PCR were 94°C for 2 min, followed by 35 cycles of 43°C for 30 sec, 72°C for 3.5 min, 94°C for 30 sec, and a final 8 min incubation at 72°C. A product with the expected size of about 2.6 kbp was amplified, double-digested with XbaI and BglII, and ligated into pVL1393 that had been digested previously with the same restriction enzymes and dephosphorylated. The entire insert was sequenced. The transfer vector was co-transfected with Baculo Gold viral DNA (PharMingen) into Spodoptera frugiperda Sf9 insect cells using lipofectin (Life Technologies). After 6 days of incubation at 27°C, cells were pelleted by centrifugation and the supernatant containing recombinant baculoviruses was used to infect Sf21 cells. After 4 days, cells were pelleted by centrifugation, washed three times with 10 m m Na-phosphate (pH 7.4), 137 m m NaCl, 2.7 m m KCl, and lysed by sonication. Cleared extracts were desalted by repeated centrifugal ultrafiltration in 50 m m Na-phosphate (pH 7.0) containing 1 m m DTT by using Centricon CC-10 ultrafiltration units (Amicon).
The authors would like to thank Dr Rainer Prohaska for protein sequencing and Barbara Svoboda for insect cell culture. T.P. wishes to thank Ullrike Fröhwein and Dr Haralt Leiter for their helpful advice. This work was supported by the Austrian Science Foundation (FWF, project P10917-BIO).
EMBL sequence database accession number Y19024(Vigna angularis stachyose synthase).