Molecular Cloning of Sucrose Isomerase Gene and Agrobacterium-Mediated Genetic Transformation of Potato (Solanum tuberosum L.) Plants

Potato (Solanum tuberosum L.) is one of the most common and important food sources on the planet, and they essential as a staple dietary item for much of the world's population. Potatoes contain carbohydrates, which lead to high blood sugar. Palatinose (isomaltulose, 6-O-alpha-D-glucopyranosyl-D-fructose) is a functional isomer of sucrose its non-cariogenicity low calorific value and it is an ideal sugar substitute to use in food production. The sucrose isomerase (palI) gene that is obtained from Erwinia rhapontici is one of the most common genes that can convert sucrose into palatinose. In present study, pQE-30- palI construct was succeffuly transformed and expression into E. coli. Sucrose isomerase (palI) gene was cloned and overexpressed into a plant expression vector pBinAR- palI contains sucrose isomerase gene (palI) fused to proteinase inhibitor II signal sequence under CaMV-35S promoter and Octopine Synthase (OCS) terminator. Expression of the protein was verified by western blot assay. Also, expression of the palI gene within the apoplast of transgenic tubers under control of a tuber-specific patatin class I B33 promoter instigated quantitative conversion of sucrose into palatinose. Tuber extracts from potato cv. Désirée were analyzed for their soluble carbohydrate composition using HPLC.


I. INTRODUCTION
Potato (Solanum tuberosum L.) is a staple food and is considered the most important economic tuber crop around the world (Joseph et al., 2015). Sucrose substitutes vary greatly in degree of sweetness, volume, texture and stability under different conditions and sweetener is not a perfect substitute for sucrose in all applications. In some cases high amounts of sucrose over long periods was found to cause cancer diseases (Price et al., 1970). Some microbes contain Isomaltulose synthase (PalI), catalyzes the isomerization of sucrose to produce isomaltulose (palatinose,-D-glucopyranosyl-1, 6-D-fructofuranose) and trehalulose (-D-glucopyranosyl-1, 1-D-fructose), as the main products with residual amounts of glucose and fructose (Börnke et al., 2001;Zhang et al., 2003;Watzlawick and Mattes, 2009). Isomaltulose is a naturally occurring isomer of sucrose (-D-glucopyranosyl-1,2-Dfructofuranose) that is valued as an acariogenic sweetener (Takazoe, 1989). Palatinose is a nutritional sugar it is digested more slowly than sucrose and has health advantages for diabeticsand nondiabetics (Lina et al., 2002). Also, it is the first non-cariogenic sugar, similar physico-chemical properties as sucrose (taste, texture, and mass) but shows a slower rate of release of monosaccharides into blood (Goda and Hosoya, 1983;Minami et al., 1990).They described the report onthe cloning and characterization of a bacterial sucrose isomerase (palI) gene from Erwinia rhapontici which catalyses the conversion of sucrose into palatinose and its function has been tested by heterologous expression in Escherichia coli. This enzyme is strictly substrate-specific toward sucrose and the reaction is essentially irreversible. The yield of palatinose formed from sucrose ranged from 85 and 15% for trehalulose, respectively (Cheetham, 1984). Expression of a chimeric sucrose isomerase (palI) gene within the apoplasm of transgenic tobacco plants and accumulated considerable amounts of non-cariogenic sucrose isomer palatinose (Xuguo et al. 2016). However, conversion of sucrose into the non-metabolizable isomer palatinose caused severe growth retardations in these plants most likely due to the depletion of a carbohydrate source for sink development (Börnke et al., 2002). In this study, description the cloningand characterization of a chimeric sucrose isomerase (palI( gene from Erwinia rhapontici and introduced palI gene with different promoter into potato plants were described.In addition to, Expression of the palI gene which conversion of sucrose into palatinose within the apoplast of transgenic tubers was studies. II. MATERIALS AND METHODS Isolation and cloning of sucrose isomerase gene. The coding region of the sucrose isomerase (palI) was cloned by polymerase chain reaction (PCR).Genomic DNA from E. rhapontici was isolated by a standard protocol and used as a template. Amplification was carried out using the following specific gene primers 5′-GGGATCCTCACCGTTCAGCAATCA3′and 5′-GTCGACCTACGGATTAAGTTTATA-3′, which were obtained from sucrose isomerase sequence (GenBank Acc. No.: AF279281) and signal peptide of proteinase inhibitor II gene (Keilet al.,1986), which was fused via a linker with the sequence ACC GAA TTG GG to the Erwinia rhapontici sucrose isomerase gene, which comprises the nucleotides 109 to 1803. Thus, a signal peptide of a plant protein, which is required for the uptake of proteins into the endoplasmic reticulum (ER) was fused N-terminally to the sucrose isomerase sequence. Plasmid Construction and bacterial strain. The Erwinia rhapontici sucrose isomerase gene and expression vector pQE-30 (Qiagen Inc. Valencia, CA) were digested with restriction enzymes BamHI/SalI respectively. The digested products were separated using agarose electrophoresis and the bands were extracted. The purified sucrose isomerase gene (palI) and the linear vector were ligated overnight at 16°C with T4 DNA ligase followed by transformed into E.coli JM109 competent cells. The transformation mixture was plated on (Luratia Broth) LB agar plates containing ampicillin (100μg/mL -1 ). The plates were incubated for 16h at 37°C. The desired recombinant plasmid pQE-30-palI was confirmed by PCR and restriction enzyme digestion with BamHI/SalI and DNA sequencing (Invitrogen). Construction of the sucrose isomerase overexpression construct the coding region of the palI gene, ranges from codons 109 to 1,803bp was amplified from Erwinia rhapontici by PCR. The palI gene fused to proteinase inhibitor II signal sequence (SP) was inserted in sense orientation between the CaMV-35S promoter and Octopine synthase (OCS) terminator within a binary vector plasmid pBinAR by the restriction endonuclease Asp718 (Höfgen and Willmitzer, 1990;Börnke et al., 2001). This plasmid is a derivative of the binary vector pBin19 (Bevan, 1984). The A. tumefaciens strain LBA4404 harbouring recombinant binary vector plasmid pBinAR-palI was maintained on LB medium (Chilton etal.,1974) supplemented with 25 mg L -1 rifampicin and 50 mg L -1 kanamycin and incubated overnight at 28°C in an incubator shaker at 90 rpm/min before using in transformation ( Fig. 1). Fig.1: Structure of palI expression construct used to transformed potato. The palI coding region ranging from 109 to 1,803bp was inserted between the CaMV-35S promoter and OCS terminating region of vector pBinAR using BamHI and Sal I restriction sites, respectively. The signal sequence for the proteinase inhibitor II (SP) was inserted in front of the palI coding region.
The 35S promoter was removed from the pBinAR vector using the restriction endonucleases EcoR I and Asp718. A fragment with a length of about 1526 by comprising the tuber-specific promoter of the class I patatin gene (B33promoter) of potato and inserted into the pBinAR vectorby EcoR I and Asp718. This resulted in the plant expression vector pBin33-Kan.The palI gene was introduced in the sense orientation into the plant transformation vector pBin33-Kan by Asp718/XbaI between the patatin B33 promotor and the octopine synthase polyadenylation signal (Fig. 2). The leaf explants were immersed in the Agrobacterium suspension containing either the binary vector pBinAR-palI or the binary vector pBin33-Kan for 20 min. Afterinfection, the leaf explants were placed on MS medium-free hormone at 25±2°C for co-cultivation of 48h. And then, the cultures were transferred on shoot induction medium (MS plus 100 mg L -1 kanamycin and 300 mg L -1 cefotaxime to inhibit further bacterial growth) for six weeks and then cultures were incubated at 25±2°C with a 16/8 h light/dark photoperiod provided by coolwhite fluorescent lamps. Shoots were transferred to MS medium 1.0 mg L -1 BAP, 0.5 mg L -1 kinetin, 10% sucrose with100 mg L -1 kanamycin and 300 mg L -1 cefotaxime at 25±2.0°C under darkfor micro tubers formation. Transformation frequency was calculated by multiplying the percentage of explants that produced plants by the percentage of plants that were confirmed to be transgenic by PCR. Expression of palI gene in E. coli. E. coli XL-1Blue cells were transformed with recombinant plasmid pQE-30-palI extracted from JM 109. The bacteria cells were grown in 5 ml LB liquid medium at 37°C with 100 μg/mL -1 ampicillin for 4-6 h at 220 rpm till to an absorbance of 0.6 at 600 nm. Expression of the fusion protein was induced by isopropyl-β-Dthiogalactopyranoside (IPTG) with a final concentration of 0.5 mM for 5 h at37°C. These samples were harvested by centrifugation at 13.000 rpm for 1min. The pellet was resuspended in 1 ml of 30 mM HEPES-KOH (pH 7.5) for preparation of palatinose. The suspension was centrifuged at 15.000 rpm for 2 min at 4°C, and the supernatant was used for enzyme measurements.The expression of palI protein was analyzed by SDS-PAGE. For conversion of sucrose into palatinose by E. Coli cell suspension, the E. coli cells (1 g wet wt.) expressing the palI gene were washed two times with 50 mmol L -1 PBS (pH 6.0). The cell pellets were resuspended in the same solution at the desired concentrations. Reactions were conducted in 50 ml flasks containing 10 ml 550 g L -1 sucrose solution at 30 °C and shaken at 150 r/min for about 5 h. Aliquots of the reaction mixture were sampled and analyzed for the amounts of palatinose formed. The reactions were terminated by heating the flasks for 10 min in a boiling water bath. Antibody preparation and western blot assay. The sucrose isomerase (palI) gene subcloned in expression vector pQE-30 which resulted pQE-palI and introduced into E. coli XL-1Blue. The protein over expressed in E. coli was purified to form antibodies against sucrose isomerase (palI)by immunization rabbit. The plant proteins were separated by SDS-PAGE and transferred to the porablot and incubated with antibodies against the palI protein from transgenic potato plants.

HPLC analysis
Reaction products were filtered through 0.22 μm membrane filters before HPLC analysis (Agilent 1200, USA system equipped with a refractive index detector). The samples were diluted 10-fold and 20 μl of diluted sample was injected onto a Rezex RCM-Monosaccharide Ca ++ column (Phenomenex, USA) for measurement of the sugar composition. The mobile phase was water with a flow rate of 0.5 ml/min at 80 °C. Glucose, fructose, sucrose and palatinose were used as standard sugars. HPLC analysis of soluble carbohydrate composition of tuber extracts was carried out as previously described (Börnke et al., 2002). The samples extracted with 80% ethanol at 80°C for one hour. Gel preparation and electrophoresis All plasmid, restriction digestion and amplified PCR products were loadedonto 1.5 % agarose gel. The purity and concentration of amplified product waschecked from the band in agarose gels. Concentration of the DNA wasestimated using a 1Kb DNA Ladder.

Statistical analysis
The experiments were three replicates for each treatment, each treatment contain 30 Jars (ten jar for each replicate), and four explants were cultured in Jar. Analysis of variance (ANOVA) was applied to data using Costat Software (2006). The differences among means for all treatments were tested for significance at 5% level by Duncan (1955).All values are reported as means ± standard deviation.
III. RESULTS AND DISCUSSION In vitro regeneration ofpotato (Solanum tuberosum L.) plants.
The study of the effect of growth regulators on the shoot induction of potato cv. Désiréeafter six weeks is shown in table 1. The highest value of mean number of shoots per explant (39.65) and mean length of shoots per explant (33.5 mm) was recorded on MS medium containing 0.1 mg L -1 IAA and 3.0 mg L -1 ZR and also this treatment had the highest regeneration rates (82 %) after six weeks compared to other treatments (Fig.3 A and B). The mean number of shoots and regeneration percentage was increased in parallel with increasing concentration of ZR up to 3.0 mg L -1 and then mean number of shoots per explant (  Microtuber formation ofpotato (Solanum tuberosum L.) cv. Désirée. The effect of sucrose on microtuber production was presented in Table 2. The results showed that the percentage of plants producing microtuber increased with the increase of sucrose concentrations. The highest number of microtuber was (5.25) detected under 10% sucrose whereas the lowest number was (2.35) at 5% sucrose. Also, the highest average weight of microtuber was (397 mg) observed on 10% sucrose as shown in Figure 4. The present investigation was tended to find out the effect of sugar level on microtuberization and found that microtuberization increased with the increasing sugar level and the optimum concentration was 10% sucrose which was similar to observed previously by Saha et al. (2013). Potato tubers are modified shoots closely associated with stolons from which they develop (Fig. 4). Tubers and stolons differ by planes of cell division which in stolons promote elongation while, in tubers increase their thickness. Signal that the plant is competent to produce tubers generated in leaves is transmitted to other plant parts by the phloem system. Signal induces a change in the plane of the cell elongation and division. Cell division plates become parallel to the elongation axis of stolons promoting radial growth. At the sub cellular level, transition in the plane of cell divisions is connected with the arrangement of microtubules (Efstathioset al., 2012). Under in vitro conditions, the change in the microtubules arrangement in the subapical zone of stolon outgrowth can be observed on the tuberization medium after 50 days (Sanz et al., 1996).
The recombinant plasmid pQE-30-palI was transformed into bacteria (JM 109)and restriction endonuclease digestion with BamH I/Sal I (Fig.5). The recombinant expression vector plasmid pQE-30-palI extracted from JM 109 and then transformed into E. coli XL-1Blue cells.To conform thegene expression of protein product, the palI gene was expressed in E. coli under the control of an IPTGinduciblepromoter.Enzymatic activity was assayed by incubation ofa crude cell extract prepared from the expressor strain withsucrose solution and by subsequent sugar analysis via HPLC.Chromatograms of bacterial extraction indicated the presence of additional peaks in the reaction mixture. In comparison with the standards, the results indicated this major peak of palatinose (Fig. 6). This clearly demonstrates the sucrose isomerase activity of PalI gene to convert sucrose to palatinose. The conversion of sucrose into palatinose drastically affected the sucrose content of E. coli. These results were agreed with (Börnke et al., 2001). The appearance of palatinose in the bacterium extract indicated the sucroseisomerase activity of the recombinant PalI proteinand the ability of the palI gene to convert sucrose to palatinose. Glucose and fructose as by-products of the reaction has been described previously (Cheetham, 1984). He also mentioned that the optimum pH for isomerase activity was between 6.0 and 6.5 and optimum temperature was 30°C, which is in good agreement with the finding that the enzyme is localized to the periplasmic space of E. rhapontici cells.   -3, Issue-3, May-June-2018  http://dx.doi.org/10.22161/ijeab/3.3.22  ISSN: 2456-1878 www.ijeab.com Page | 870 Transformation of potato (Solanum tuberosum L.)cv.

Désirée. Binary vectorspBinAR-palIand pBin33-Kan.
A. tumefaciens colonies transformed with pBinAR-palIor pBin33-Kan wereanalyzed by colony PCR using palIgene-specific primers. The expectedband size of 1803 bp was observed in selected colonies. This confirmed the presence of the vector in the colonies.Presence of the pBinAR-palIor pBin33-Kan plasmid was verified in PCRpositive colonies by digestion with Asp718/XbaI. A 1804 bp fragment wasreleased from DNA extracted from the colonies (Fig.7). These results confirm successful cloning of the expression vector pBinAR-palIor pBin33-Kan into A. tumefaciens.

Agrobacterium-mediated transformation.
Leaves excised from 4 to 6 week-old in vitrogrown potatocv. Désiréeas explants and incubated with Agrobacterium strain LBA4404 carrying either the binary vector pBinAR-palI or the binary vector pBin33-Kan for 20 min. Explants were co-cultivated with Agrobacterium for 48 h on the MS medium-free of hormones. After cocultivation in the dark, the explants were transferred to the shoot induction medium containing 0.1 mg L -1 IAA and 3.0 mg L -1 ZR with 300 mg L -1 cefotaxime and 100 mg L -1 kanamycin to select for transformed cells for six weeks. And then cultures were incubated at 25±2°C with a 16/8 h light/dark photoperiod provided by cool-white fluorescent lamps. Shoots were transferred to a rooting medium containing 1mg L -1 IBA and 50 mg L -1 kanamycin and then the plantlets were transferred to greenhouse and grown in soil. At this time, the transformation efficiency was evaluated for further analysis with Agrobacterium harboring the binary vector pBinAR-palI. For in vitro tuber formation, the transgenic potato shoots were cultured on MS medium containing 1.0 mg L -1 BAP, 0.5 mg L -1 kinetin, 10% sucrose with 50 mg L -1 kanamycin and then shoots were incubated under dark condition at 25±2.0°C. Transgenic potato plantlets were checked by PCR and Western analyses. Potato leaf discs were transformed using Agrobacterium-mediated gene transfer under patatin promoter gives rise to tuber-specific expression (Rocha-Sosa et al., 1989). This result is also in accordance with Sarker and Mustafa (2002) where histological GUS assay showed the expression of GUS gene in the leaf tissues of transformed shoots. While, recent study, Veale et al. (2012) used In vitro potato explants were infected with Agrobacterium LBA4404 strain harbouring the binary vector pSPUD5 carrying the cry1Ia1 gene under the transcriptional control of the (ocs) promoter and the nptII gene, cultured on the pre-culture medium with 50 μM acetosyringone.

PCR-detection
To confirm the stable transformation in the genome, DNA was isolated using genomic DNA isolation kit (Bio Basic, Canada) from putative transformed plants of high dose (100 mg L -1 ) kanamycin exposure plants. After transformation with Agrobacterium strain LBA4404 carrying either the binary vector pBinAR-palI or the binary vector pBin33-Kan contains sucrose isomerase gene (palI) fused to the signal peptide of proteinase inhibitor II gene (SP), isolated DNA was quantified and amplified by PCR using the specific primers. The quality of the plant DNA was confirmed by a positive control PCR reaction.
Sixty putatively transformed plants were tested for palI gene. PCR analysis revealed that putative potato transformed plants displayed expected 1803 bp size band (size 1694 bp of palI gene and size 109 bp of signal peptide of proteinase inhibitor II gene) as shown in (Fig.8) et al., 2002). 60 regenerated potato shoots were screened for expression of palI protein in vitro plantlets by Western Blotting assay using a polyclonal antibody raised examined leaves of the palI-expressing potato. Total proteins were descriptively isolated by SDS-PAGE and blotted the protein with antibodies specific for palI-proteins. Our results showed that palI protein was expressed in all samples of transgenic potato plants compared to the nontransgenic potato control. This is in contrast to results obtained with constitutive expression of palI in transgenic potato plants. The detection of palIprotein can be performed using any part of potato plantlets, which was expressed mainly under the regulation of the CaMV-35S promoter. In addition the antibody obtained will provide a basic detector of genetically modified potato plants. Rui-juanet al. (2016) used western blot assay for detection PMI protein in genetically modified rice and showed that PMI was expressed in all samples except anther, indicating the constitutive expression of PMI in genetically engineered rice.

HPLC analysis
Sucrose isomerase expression under the control of the tuber-specific patatin class I B33 promoter leads to in vivo conversion of sucrose into palatinose, tuber extracts from potatocv. Désiréewere analyzed for their soluble carbohydrate composition using HPLC. The chromatograms assay indicated that there was an additional major peak that was not present in the control and compared to the standard this peak could be set to palatinose (Fig.9). An additional minor peak eluted close to the sucrose signal in extracts from transgenic tubers.Quantitative analysis of non-structural carbohydrates of transgenic tubers has shown accumulation of palatinose in the range of 2.4 mol g -1 FW to 19.5 mol g -1 FW while, sucrose and glucose content was only 1.4 mol g -1 FW and 0.48 mol g -1 FW, respectively, whereas the non-transgenic tubers contained 16.8 mol g -1 FW sucrose and 4.85 mol g -1 FW glucose. The conversion of sucrose into palatinose drastically affected the sucrose and glucose content of transgenic tubers. These results indicate almost quantitative conversion of sucrose into palatinose via palI expressing potato tubers. These results are compatible withRocha-Sosa etal. (1989), they have the sucrose isomerase gene expression under the control of the tuber-specific patatin class I B33 promoter in transgenic potato plants. This protein was combined with the signal peptide of the proteinase inhibitor II, which governs secretion of the enzyme into the apoplasmic space (Von Schaewen et al., 1990).The patatin class I B33 promoter it seems inactive during early tuberization (Tauberger et al., 1999). The apoplasmic localization of the sucrose isomerase leads to accumulation of palatinose. This indicates the presence of sucrose within the apoplast even in later stages of tuber development which have been interpreted as a result of