Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Recombinant protease inhibitors represent useful tools for the development of insect‐resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad‐spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI‐expressing and control (comparator) lines, followed by a two‐dimensional gel electrophoresis (2‐DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI‐expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2‐DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2‐DE showed unaltered deposition patterns in SlCDI‐expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI‐expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel‐generation transgenic plants.     

Potato flour viscosity improvement is associated with the expression of a wheat LMW-glutenin gene

It has been previously shown that expression of a high-molecular-weight glutenin (HMW-GS) in transgenic wheat seeds resulted in the improvement of flour functional properties. In this study, potato flour viscosity was improved through a specific expression of a low-molecular-weight glutenin (LMW-GS-MB1) gene in tuber. The resulting construct was introduced into potato leaf explants (Solanum tuberosum cv Kennebec) through Agrobacterium tumefaciens-mediated gene transfer. Southern and Northern analysis of transgenic potato confirmed that the integration of LMW-GS-MB1 in genomic DNA was stable and its mRNA was abundant in transgenic line 16 tubers. Western blot analysis of line 16 extract shows a LMW-GS subunit accumulation in tuber. To demonstrate the capacity of transgenic lines to produce tubers with improved flour functional properties, transgenic lines 9 and 16 exhibiting, respectively, moderate and high expression of LMW-GS-MB1 mRNA and nontransgenic plants were transferred to field plots. The mean viscosity value of flour obtained from the field-grown tubers of transgenic line 16 exhibited a 3-fold increase in viscosity at 23 degrees C when compared to flour from nontransgenic tubers.     

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.     

Detecting a difference – assessing generalisability when modelling metabolome fingerprint data in longer term studies of genetically modified plants

There is current debate on whether genetically-manipulated plants might contain unexpected, potentially undesirable, changes in overall metabolite composition relative to that of the progenitor genotype. However, appropriate analytical technology and acceptable metrics of compositional similarity require development, particularly to allow data integration from different laboratories and different harvests. For an initial comprehensive overview of compositional similarity, we explored the use of a rapid and relatively non-selective fingerprinting technique based on flow injection electrospray ionisation mass spectrometry (FIE-MS). Six conventionally-bred potato cultivars and six experimental bioengineered potato genotypes were produced in four field blocks during two growing seasons and analysed on two different analytical instruments (LCT, Micromass in 2001 and LTQ, Thermo Finnigan in 2003). Field effects and overall process variability was found to be negligible when compared to inherited genotype variance. The data derived separately for experiments using tubers from individual harvest years were compared to assess the generalisability of models for the comparison of GM and non-GM potato tubers under investigation. This procedure proved appropriate for not only rapid assessment of similarities between plant genotypes but also to predict the identity of metabolite signals that could explain differences between genotype classes irrespective of the instrument used for analysis. Importantly, despite differences in ionisation and data acquisition properties of the two instruments the generalisation of models could be confirmed after correlation analysis of explanatory variables correctly identified the molecular origin of differences between genotypes. We conclude that FIE-MS metabolomics fingerprinting technology coupled to machine learning data analysis has great potential as a robust tool for first-pass metabolic phenotyping and, therefore, initial assessments of compositional similarities prior to use of more targeted hyphenated gas or liquid chromatography-mass spectrometry techniques.     

Expression of antibodies and Fab fragments in transgenic potato plants: a case study for bulk production in crop plants

To use transgenic potato tubers (Solanum tuberosum cv. Désirée) for bulk production of recombinant antibodies, constructs were engineered for accumulating full-size IgGs and Fab fragments in the plant cell apoplast or endoplasmic reticulum (ER). An in-house transformation protocol was worked out for the efficient co-transformation of potato root explants. Accumulation levels in tubers of up to 0.5% of total soluble protein were found for antibodies targeted to the ER whereas five-fold lower accumulation levels were found for antibodies targeted for secretion. Additionally, different aspects important for the commercial exploitation of potato tubers as a heterologous production system were analysed. Tubers could be stored for up to 6 months without significant loss of antibody amount or activity. Minor variations in antibody accumulation levels were observed in tubers that originated from the same transformant. Most isolated IgGs and Fab fragments bound the antigen and had the correct molecular weight when compared with the hybridoma-derived standard. Processing to greenhouse or field trials, including in vitro propagation of a selected transformant, required only approximately 9 months from the start of transformation, a time frame in which hundreds of kilograms of transgenic potato tubers could easily be obtained. Small-scale purification of IgG was possible by using standard laboratory techniques. Thus, molecular farming in potato tubers can be a viable production system for economic production of clinically or industrially interesting macromolecules, such as antibodies.     

Expression of a deregulated tobacco nitrate reductase gene in potato increases biomass production and decreases nitrate concentration in all organs

We investigated the physiological consequences for nitrogen metabolism and growth of the deregulated expression of an N-terminal-deleted tobacco nitrate reductase in two lines of potato (Solanum tuberosum L. cv Safrane). The transgenic plants showed a higher biomass accumulation, especially in tubers, but a constant nitrogen content per plant. This implies that the transformed lines had a reduced nitrogen concentration per unit of dry weight. A severe reduction in nitrate concentrations was also observed in all organs, but was more apparent in tubers where nitrate was almost undetectable in the transgenic lines. In leaves and roots, but not tubers, this nitrate decrease was accompanied by a statistically significant increase in the level of malate, which acts as a counter-anion for nitrate reduction. Apart from glutamine in tubers, no major changes in amino acid concentration were seen in leaves, roots or tubers. We conclude that enhancement of nitrate reduction rate leads to higher biomass production, probably by allowing a better allocation of N-resources to photosynthesis and C-metabolism.Abbreviations DAP Days after planting - Gln Glutamine - NR Nitrate reductase - WT Wild type     

Comparison of tuber proteomes of potato varieties, landraces, and genetically modified lines

Crop improvement by genetic modification remains controversial, one of the major issues being the potential for unintended effects. Comparative safety assessment includes targeted analysis of key nutrients and antinutritional factors, but broader scale-profiling or "omics" methods could increase the chances of detecting unintended effects. Comparative assessment should consider the extent of natural variation and not simply compare genetically modified (GM) lines and parental controls. In this study, potato (Solanum tuberosum) proteome diversity has been assessed using a range of diverse non-GM germplasm. In addition, a selection of GM potato lines was compared to assess the potential for unintended differences in protein profiles. Clear qualitative and quantitative differences were found in the protein patterns of the varieties and landraces examined, with 1,077 of 1,111 protein spots analyzed showing statistically significant differences. The diploid species Solanum phureja could be clearly differentiated from tetraploid (Solanum tuberosum) genotypes. Many of the proteins apparently contributing to genotype differentiation are involved in disease and defense responses, the glycolytic pathway, and sugar metabolism or protein targeting/storage. Only nine proteins out of 730 showed significant differences between GM lines and their controls. There was much less variation between GM lines and their non-GM controls compared with that found between different varieties and landraces. A number of proteins were identified by mass spectrometry and added to a potato tuber two-dimensional protein map.     

Comparison of transgenic <Emphasis Type="Italic">Gerbera hybrida</Emphasis> lines and traditional varieties shows no differences in cytotoxicity or metabolic fingerprints

Genetic modification using gene transfer (GM) is still controversial when applied to plant breeding at least in Europe. One major concern is how GM affects other genes and thus the metabolism of the plant. In this study, 225 genetically modified lines of the ornamental plant Gerbera hybrida and 42 non-GM gerbera varieties were used to investigate changes in secondary metabolism. The cytotoxicity of GM and non-GM gerbera extracts was evaluated on human cell lines derived from lung, liver, and intestinal tissues. The results indicate that the safety profile for GM gerbera lines is similar to the viability pattern for non-GM varieties-none of the extracts were toxic. In addition, metabolic fingerprints of gerbera extracts were identified using thin-layer chromatography and analysed by principal component analysis (PCA), the nearest neighbour classifier, and Fligner-Killeen test. No new compounds unique to GM lines were observed. With PCA, no separation between GM gerbera lines and varieties could be demonstrated. In the nearest neighbour classifier, 54% of the samples found the expected neighbour based on the gene constructs used for transformation. With Fligner-Killeen test, we studied if the amounts of compounds vary more in GM gerberas than in varieties. In most cases, there were no statistically significant differences between the varieties and GM lines or there was more variation among the non-GM varieties than in the GM lines. The variance of a single compound was significantly larger in transgenic gerbera lines than in varieties and of three compounds in non-GM varieties.     

Resistance of potatoes transgenic for a cry1Ac9 gene, to Phthorimaea operculella (Lepidoptera: Gelechiidae) over field seasons and between plant organs

Stable performance of insect‐resistant transgenic plants across field seasons and between plant organs damaged by the insect pest is critical for management of this resistance in the field. To evaluate this, potato (Solanum tuberosum) lines transgenic for a cry1Ac9 gene with resistance to potato tuber moth (Phthorimaea operculella) were established in the field during the southern hemisphere summers of 1997/98, 1998/99 and 1999/00 as small field plots, each of 10 plants. Replicate plots of the non‐transgenic parent cultivars (at least one for every three independently derived transgenic lines) were planted randomly throughout the trials. Field‐grown foliage was challenged with larvae in the laboratory and a growth index (GI) was calculated for recovered larvae from each transgenic and non‐transgenic potato line. Larval growth on young and mature leaves, and on newly harvested or stored tubers was also measured in the laboratory. Foliage from the transgenic lines inhibited larval growth in all seasons tested. For both control and transgenic lines, larvae had slightly lower GIs when reared on mature leaves compared with young leaves, although the correlation between mean GI for young and mature transgenic leaves was high (r = 0.97). The correlation between the mean GIs of larvae on newly harvested tubers and on those stored for 5 months was also high (r = 1.0). However, the GIs of larvae on newly harvested transgenic tubers were larger than on transgenic tubers stored for 5 months. The relative growth indices (RGI = mean GI/number days before final weighing) of larvae reared on newly harvested tubers from transgenic lines were generally higher than those from young transgenic foliage, while the RGIs of larvae reared on non‐transgenic tubers were slightly lower than those fed non‐transgenic foliage. The correlation between mean RGIs of larvae fed tubers or foliage was 0.62. The transgenic potato lines exhibited stable resistance to larvae across field seasons, between affected plant organs, and between plant organs of different ages.     

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229–1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and ¹³C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyrophosphorylase antisense plants was observed.Abbreviations CaMV cauliflower mosaic virus - NMR nuclear magnetic resonance We gratefully acknowledge Dr. Ulrich Eder (Schering AG, Berlin, Germany) for performing ¹³C-NMR spectroscopy, and Dr. Susanne Hoffmann-Benning (Institut für Genbiologische Forschung) for introducing us to immunohistochemistry. We thank Jessyca Dietze for plant transformations, Birgit Burose for taking care of greenhouse plants, and Antje Voigt for photographic work.     

Stable transformation and tissue culture response in current European winter wheats (Triticum aestivum L.)

In order to efficiently complement traditional wheat breeding with genetic transformation technology it will be desirable to introduce transgenes into the ideal genetic background. Poor tissue culture performance is limiting the number of wheat genotypes that can be stably transformed. We statistically analysed the tissue culture response of 38 current European winter wheats and discuss genetic factors influencing this trait. Although regenerable callus cultures could be initiated from immature embryos of all 38 winter wheats analysed, the number of regenerated plants per cultured explant differed highly significantly (p<0.01) among genotypes. Ten cultivars with excellent ranking in this parameter were selected for transformation experiments. Independent transgenic plants were recovered from nine winter wheat genotypes with a frequency ranging between 0.2% and 2.0% of the cultured immature embryos after biolistic transfer of the bar gene and bialaphos selection. The nine transformable winter wheat genotypes included a recently released high-yielding, disease-resistant cultivar (cv. Certo), well established cultivars with elite bread-making quality (cv. Tarso, Alidos) and current breeding lines differing in yield, disease resistance and grain quality. Transgene integration and expression were confirmed by Southern blot analysis, polymerase chain reaction, phosphinothricin acetyltransferase activity assay and herbicide application. Transgene expression was stably transmitted to the sexual progeny of all transgenic lines analysed and segregated in a Mendelian fashion in the majority of lines. The introduction of transgenes into the ideal genetic background will allow a thorough evaluation of their crop improvement potential.     

Increased fatty acid production in potato by engineering of acetyl-CoA carboxylase

In contrast to oil seeds, potato (Solanum tuberosum L.) is characterized by a high amount of starch stored in the tubers. To assess the capacity for oil synthesis in potato tubers, the changes in lipid content and flux into lipid synthesis were explored in transgenic potatoes altered in carbohydrate or lipid metabolism. A strong decrease in the amount of starch observed in antisense lines for ADP-glucose pyrophosphorylase or plastidic phosphoglucomutase had no effect on storage-lipid content. Similarly, potato lines over-expressing the Arabidopsis thaliana (L.) Heynh. plastidic ATP/ADP transporter that contained an increased amount of starch were not altered in oil content, indicating that the plastidic ATP level is not limiting fatty acid synthesis in potato tubers. However, over-expression of the acetyl-CoA carboxylase from Arabidopsis in the amyloplasts of potato tubers led to an increase in fatty acid synthesis and a more than 5-fold increase in the amount of triacylglycerol. Taken together, these data demonstrate that potato tubers have the capacity for storage-lipid synthesis and that malonyl-CoA, the substrate for elongation during fatty acid synthesis, represents one of the limiting factors for oil accumulation.Abbreviations AATP Plastidic ADP/ATP transporter - ACCase Acetyl-CoA:carboxylase - DGAT Acyl-CoA:diacylglycerol acyltransferase - FW Fresh weight - TLC Thin-layer chromatography - WT Wild typeSource for transgenic plant material. Upon request, transgenic potato lines altered in ACCase activity can be obtained from Peter Dörmann. For potato lines with alterations in AATP transporter activity, please refer to H. Ekkehard Neuhaus. Transgenic AGP and PGM lines are available from A. Fernie (Max-Planck-Institute of Molecular Plant Physiology, Golm, Germany).     

Over-expression of strawberry d-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance

Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed transgenic potato plants (Solanum tuberosum L. cv. Taedong Valley) over-expressing strawberry GalUR gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the GalUR gene in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid (AsA) levels in transgenic tubers were determined by high-performance liquid chromatography (HPLC). The over-expression of GalUR resulted in 1.6–2-fold increase in AsA in transgenic potato and the levels of AsA were positively correlated with increased GalUR activity. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen (MV), NaCl or mannitol as compared to untransformed control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of GalUR gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control.     

Nutritional properties of tubers of conventionally bred and transgenic lines of potato resistant to necrotic strain of Potato virus Y (PVYN)

The potential effect of genetic modification on nutritional properties of potatoes transformed to improve resistance to a necrotic strain of Potato virus Y was determined in a rat experiment. Autoclaved tubers from four transgenic lines were included to a diet in the amount of 40% and compared with the conventional cv. Irga. The experiment lasted 3 weeks and special attention was paid to nutritional properties of diets, caecal metabolism and serum indices. Genetic modification of potato had no negative effect on the chemical composition and nutritional properties of tubers, ecosystem of the caecum, activity of serum enzymes and non-specific defence mechanism of the rats. Obtained results indicate that transgenic potato with improved resistance to PVY(N): line R1F (truncated gene coding for PVY(N) polymerase in sense orientation), R2P (truncated gene coding for PVY(N) polymerase in antisense orientation), and NTR1.16 (non-translated regions of PVY(N) genome in sense orientation) are substantial and nutritional equivalence to the non-transgenic cultivar. Tubers of transgenic line NTR2.27 (non-translated regions of PVY(N) genome in antisense orientation) increased the bulk of caecal digesta and the production of SCFA as compared to tubers of the conventional cultivar and the other transgenic clones. Taking into account some deviations, it seems reasonable to undertake a long-term feeding study to confirm the nutritional properties of tubers of transgenic lines.     

Production of high-starch, low-glucose potatoes through over-expression of the metabolic regulator SnRK1

Transgenic potato ( Solanum tuberosum cv. Prairie) lines were produced over-expressing a sucrose non-fermenting-1-related protein kinase-1 gene ( SnRK1 ) under the control of a patatin (tuber-specific) promoter. SnRK1 activity in the tubers of three independent transgenic lines was increased by 55%−167% compared with that in the wild-type. Glucose levels were decreased, at 17%−56% of the levels of the wild-type, and the starch content showed an increase of 23%−30%. Sucrose and fructose levels in the tubers of the transgenic plants did not show a significant change. Northern analyses of genes encoding sucrose synthase and ADP-glucose pyrophosphorylase, two key enzymes involved in the biosynthetic pathway from sucrose to starch, showed that the expression of both was increased in tubers of the transgenic lines compared with the wild-type. In contrast, the expression of genes encoding two other enzymes of carbohydrate metabolism, α-amylase and sucrose phosphate synthase, showed no change. The activity of sucrose synthase and ADP-glucose pyrophosphorylase was also increased, by approximately 20%–60% and three- to five-fold, respectively, whereas the activity of hexokinase was unchanged. The results are consistent with a role for SnRK1 in regulating carbon flux through the storage pathway to starch biosynthesis. They emphasize the importance of SnRK1 in the regulation of carbohydrate metabolism and resource partitioning, and indicate a specific role for SnRK1 in the control of starch accumulation in potato tubers.     

Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics

ABSTRACT: BACKGROUND: The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes beta-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. RESULTS: The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. CONCLUSIONS: Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.     

人乳铁蛋白在转基因马铃薯块茎中的表达

人乳铁蛋白(human lactoferrin,hLF)是人体非特异性免疫系统的重要成员之一,具有抗细菌、真菌和抗病毒活性及其他多种功能.报道将hLF基因的cDNA与马铃薯(Solarium tuberosum L.)块茎专一性表达patain基因启动子融合后通过农杆菌介导导入马铃薯,PCR检测证实获得了多个转基因株系,RT-PCR阳性结果说明hLF mRNA在马铃薯植株中得到了表达.同时,经过ELISA及Western blot检测证实,转基因马铃薯表达了hLF并具有人乳铁蛋白的活性.