BACKGROUND
the potato  is the world's fourth most important crop.
potato tuber proteins are categorized into three groups: patatins, protease inhibitors, and other proteins  <cit> .
patatins are a family of glycoproteins and represent up to 40% of the total soluble protein in potato tubers  <cit> .
patatins accumulate in the vacuoles of tubers and leaves and are mainly found in parenchyma cells of potato tubers  <cit> .
two classes of patatin gene families have been identified in potato plants  <cit> .
class i transcripts, which lack the  <dig> nucleotides in the 5'-untranslated region , were found to be tuber specific, whereas class ii transcripts, which bear the  <dig> nucleotides, were found in both tubers and roots.
patatin genes are strongly expressed in tubers but occur at very low levels in other tissues  <cit> .
patatins are approximately 40– <dig> kda in size and are the major storage protein of potato tubers  <cit> .
the roles of patatins are proposed to include fatty acid esterase, lipid acyl hydrolase, and acyltransferase activities  <cit> .
patatins are also known to have antioxidant activities  <cit> .
however, the physiological functions of patatins in potato tubers are not fully understood.
potato tubers have been used as a plant host for the production of recombinant human proteins and vaccines  <cit> .
a study of transgenic potato plants expressing a human serum albumin gene revealed that recombinant albumin accumulated up to  <dig> % of total soluble tuber protein in the plants  <cit> .
the low level of target protein expression is a barrier to recombinant protein production in plants.
in addition, the steps involved in purification of target proteins are major cost factors in plant-based protein production  <cit> .
the phenomenon of gene silencing was first observed in the petunia  <cit> .
gene silencing is a eukaryotic genome defense system against viruses and mobile dna elements that works by processing double-stranded rna  into short interfering rna   <cit> .
rna interference  refers to a multi-step process, including the introduction of double-stranded rna  into a cell, cleavage of dsrna into short interfering rna  21– <dig> nt in size, formation of rna-induced silencing complex , degradation of complementary mrna, and suppression of target gene expression .
rnai technology has become a powerful tool for the study of the functions of individual genes in a range of organisms  <cit> .
hairpin rna , a class of short interfering rna, is composed of two target sequences in an inverted repeat orientation with a spacer between.
the two target sequences form a duplex rna with a loop  <cit> .
hprna interference  has proven to be an effective tool for the study of the functions of several genes, including the pigment biosynthesis gene chalcone synthase , the ethylene-signaling gene , and the flowering repression gene  in arabidopsis  <cit> .
in this study, we utilized the hprnai-based reverse genetic approach to knock down various patatin isoforms in potato tubers.
the objective of this study was to provide patatin knockdown potato tubers as a heterologous expression system for making human-like glycoproteins.
RESULTS
construction of a patatin-specific hairpin rnai  vector and potato transformation
prior to the construction of an hprnai vector for suppression of patatin expression, the sequences of  <dig> known genes in the patatin multi-gene family in the potato were downloaded from the ncbi database and aligned for a thorough comparison of the genes.
great similarities  between the patatin genes were found to exist at the nucleotide level .
a highly conserved  <dig> nt fragment of pat3-k <dig> , nucleotides 188– <dig>  was pcr amplified using desiree as template.
half of the pcr fragments were inserted into the xhoi – kpni site of pkannibal vector in the sense orientation and the other half of the pcr fragments were inserted into the xbai – clai site in the antisense orientation.
the patatin-hprnai frame in pkannibal was confirmed by restriction analysis and nucleotide sequencing.
the pkannibal vector was digested with noti enzyme.
noti fragments were cloned into part <dig> binary vector, resulting in a patatin-specific hprnai vector, which was driven by camv 35s promoter .
the patatin hprnai binary vector was transformed into potato plants  via agrobacterium-mediated transformation.
ten independent patatin-hprnai potato lines were selected on kanamycin.
the transgene in the hprnai lines was confirmed by pcr screening with npt ii primers  and camv 35s primers .
all ten of these lines had the npt ii gene and were resistant to kanamycin.
seven of the regenerated kanamycin-resistant potato plants were further analyzed by southern blot to determine the number of t-dna inserts .
patatin hprnai lines  <dig>   <dig>   <dig>  and  <dig> each had one t-dna insert.
analysis of patatins on 1d sds-page gel
to determine the relative efficacy of gene silencing between patatin hprnai lines, one-dimensional  sds-page analysis was initially performed using total soluble protein extracted from two month old mature potato tubers of the hprnai and wt plants grown in greenhouse.
as expected, the total protein of wt plants showed a thick band corresponding to patatin  on the sds-page gel .
to our surprise, except for line  <dig>  the patatin hprnai lines did not show patatin bands on the gel.
the patatin level in hprnai line  <dig> was similar to the wt plants.
based on the amounts of patatins, gene silencing seemed to be highly effective in the individual lines where the patatin-hprna was expressed.
these data indicate that the inverted repeat sequence of partial patatin gene pat3-k <dig>  effectively induces gene silencing.
analysis of patatin variants on 2d page gel
based upon the levels of patatin in the 1d gel, we chose four patatin knockdown lines  for further analysis of various patatin variants using two-dimensional  page gel electrophoresis.
our 2d gel analysis of wt and line  <dig> mature tuber protein confirmed that the presumed multiple patatin spots  <cit>  at  <dig> kda had disappeared.
other patatin hprnai lines  showed patterns in patatin reduction similar to line  <dig> .
overall, 2d gel analysis of the patatin-hprnai line  <dig> revealed that the number and size of patatin spots significantly reduced, whereas other protein spots almost remained unchanged.
analysis of pat3-k <dig> mrnas in hprnai lines using real-time rt-pcr
to confirm the data obtained from 1d sds-page and 2d page gel electrophoresis, a real-time rt-pcr was performed using total rna extracted from mature potato tubers  of ten independent patatin-hprnai lines grown in greenhouse.
we used a primer pair designed against the most highly conserved sequence in the patatin gene family.
the levels of various patatin transcripts present in hprnai lines were measured by a quantitative real-time rt-pcr with the primers.
as shown in figure  <dig>  real-time rt-pcr revealed that patatin transcripts in ten patatin-hprnai lines were significantly reduced.
patatin hprnai lines  <dig>   <dig>   <dig>   <dig> and  <dig> showed significant reductions  in patatin transcripts compared to wt plants.
these data are consistent with the data obtained from 1d and 2d gel analysis.
the expression of the patatin-hprna presumably led to effective inhibition of the expression of the pat3-k <dig> gene and other members of the gene family.
in vitro- and ex vitro-grown patatin-hprnai lines showed a normal growth pattern and phenotype
to examine the effects of gene silencing on the phenotype, tuber number, and average tuber weight of patatin-hprnai potato plants, the transgenic potato plants were cultured in vitro and also grown in a greenhouse.
during in vitro culture, the transgenic potato plants showed a normal morphologic characteristic and growth pattern in patatin hprnai lines  <dig>   <dig>   <dig>   <dig> .
ten patatin-hprnai greenhouse-grown potato plants were harvested and the number of tubers per potato plant and average tuber weight were compared with wild-type .
the number of tubers in patatin hprnai lines ranged from  <dig>  to  <dig> , whereas that of wild-type plants was  <dig> .
average tuber weight in patatin hprnai lines and wild-type was variable between the plants.
though average tuber weight of patatin hprnai lines seemed to be a bit smaller than that of wild-type, there were no significant differences  .
overall, patatin-knockdown potato plants showed a normal growth pattern and phenotype during in vitro culture and greenhouse cultivation.
patatin-knockdown potatoes reduced additional purification step of glycoproteins
patatin-knockdown potatoes are likely to have a few advantages in terms of glycoprotein purification, compared to wild-type potatoes.
to confirm the possible advantages of patatin-knockdown potatoes, potato glycoproteins were purified by affinity chromatography on sepharose-coupled concanavalin a  and the purified glycoproteins were monitored by running a sds-page gel .
the content of total soluble proteins was not reduced in transgenic plants, though patatin was almost completely knocked down.
in our purification system, the yields of glycoproteins were  <dig> % ,  <dig> %  and  <dig> %  .
the level of glycoproteins in wt was higher than transgenic plants.
in addition, sds-page analysis showed that the fraction of purified glycoproteins in wild-type potatoes has two major bands .
one was patatin and the other was a band in size 26– <dig> kda.
in contrast, patatin-rnai lines had only one major band .
therefore, no further purification steps to remove patatin were required.
discussion
patatins are considered to be a major storage protein in potato tubers  <cit> .
here we report that by suppressing the expression of the potato patatin multi-gene family, transgenic potato tubers accumulated dramatically low levels of patatins.
this was achieved by rna interference  that was first discovered in caenorhabditis elegans  <cit> .
in the present study, we successfully employed rnai technology to produce patatin knockdown/free potato tubers to eventually provide potato tubers as a heterologous expression system for the production of human therapeutic glycoproteins.
the potato genome  has 64– <dig> patatin dna copies, and most of the dna copies do not show much allelic variation based on restriction fragment length polymorphism  mapping  <cit> .
a study of patatins present in cv.
kuras showed that patatins are 84–96% identified at amino acid sequence levels  <cit> .
when kuras tuber protein spots from coomassie brilliant blue-stained 2d gels were assigned using tryptic pmf analysis, the major acidic protein spots, 40– <dig> kda in size, were patatin variants, and more than  <dig> patatin spots were observed on the 2d gel  <cit> .
all known potato patatins are grouped into a large family of multi-genes sharing 80–95% identity at the nucleotide level and clustered into three clades including pat <dig>  pat <dig>  and pat <dig>  <cit> .
when we aligned nucleotide sequences of  <dig> patatin genes from kuras and kennebec, patatins of both cultivars had more than 90% identity.
thus, rnai appeared to be a suitable reverse genetic approach for producing patatin-knockdown potato tubers.
based upon sequence similarity within  <dig> potato patatin genes available from ncbi, a partial cdna  of pat3-k <dig> was chosen as a target sequence to silence patatin multi-genes of desiree.
ten independent patatin-hprnai lines were generated.
in order to test the effect of gene silencing of patatins, the total soluble protein extracted from patatin hprnai lines was analyzed on 1d sds-page gel.
patatin bands were detected in line  <dig> but not in nine other hprnai lines .
since patatins account for ~40% of total soluble protein in potato tubers  <cit> , the highly reduced patatin content provided an easy measurement of the degree of gene silencing in the individual hprnai lines.
the observed suppression of patatins in patatin hprnai lines was analyzed in more detail using two-dimensional  gel electrophoresis.
2d gel of wt plants showed typically thick/various patatin spots of  <dig> to  <dig> kda, whereas major protein spots were not observed in the gel of hprnai line  <dig> .
based upon the measurement of the relative patatin content between the hprnai line  <dig> and the wt plant, patatins in line  <dig> were reduced by about 99%.
the results of 2d gel analysis suggested that individual genes of the patatin multi-gene family were significantly knocked down by rna interference.
a study of patatin antisense transgenic potato plants showed that the amount of patatin proteins in the plants is maximally reduced by 75–90%  <cit> .
our 2d gel analysis showed that patatin hprnai suppressed up to 99% of potato patatins.
the data demonstrated that the rnai approach seems to be much more effective than the antisense rna approach.
in addition to the significant reduction of potato patatins, the level of patatin transcripts was examined by a quantitative real-time rt-pcr.
the levels of patatin transcripts were dependent on the lines.
the maximum reduction of 99% in the transcript level of pat3-k <dig> was observed in line  <dig>  in comparison with that of the control pcr .
the decreased levels of transcripts in patatin-hprnai line  <dig> were significantly consistent with the suppression of patatins .
since primers of real-time rt-pcr were designed at the highly conserved gene , the dramatic reduction in pat3-k <dig> transcripts probably includes the knockdown of other patatin genes as well.
furthermore, based upon the fact that various patatin spots were not observed in the 2d gel of patatin hprnai line  <dig>  the transcripts of most patatin genes would be effectively degraded by rna interference.
overall, the maximum level of reduction in patatin rnas and proteins in the hprnai lines was approximately 99%.
human-like glycoproteins have been produced in plant systems, including potato and tobacco  <cit> .
use of more advantageous protein expression systems is necessary for plant-based human-like glycoprotein production  because the yield of recombinant proteins is very low and because expensive purification steps are required.
patatin knockdown potatoes could be used as a suitable expression host for the production of human-like glycoproteins, since very low endogenous glycoprotein patatins might make a relatively selective purification of human-like glycoproteins easier, with less patatin contamination .
furthermore, the use of patatin tuber specific promoter and n-terminal targeting sequences could easily drive the expression of human glycoproteins in the vacuoles of patatin knockdown potato tubers.
glycosylation, the attachment of oligosaccharides to proteins, is one of the major posttranslational modifications of proteins in plants and animals.
in most therapeutic glycoproteins, the sugar is attached to the asn residue of a tripeptide asn-x-ser/thr of the protein and/or thr or ser residues in the peptide backbone .
the processing of the n-linked glycans occurs in er and golgi apparatus.
plant and mammalian n-glycan maturations differ only in the late golgi apparatus .
in terms of the production of human therapeutic glycoproteins in plant systems, it is necessary to inhibit undesirable plant-specific posttranslational modifications in plants and to glycosylate target proteins in a manner similar to human systems via genetic engineering .
therefore, the first prerequisite for the production of human therapeutic glycoproteins in potato tubers is to suppress the expression of major glycoprotein "patatins".
thus, the patatin-knockdown potato tuber would be a practical candidate to host the selective production of human therapeutic glycoproteins.
CONCLUSIONS
rna silencing technology was used to produce patatin-knockdown potato tubers.
a target sequence  of highly conserved gene  in the patatin multi-gene family was amplified for the construction of a patatin-specific hairpin rnai vector.
the effects of gene silencing in patatin-hprnai lines were analyzed at the protein level by both 1d and 2d page gel electrophoresis.
in addition, the gene silencing effects were confirmed at the mrna level by a quantitative real-time rt-pcr.
dependent on the patatin hprnai line, almost all of the individual patatin variants  were reduced by approximately 99% at both the protein and mrna levels in line  <dig>  despite a significant reduction of patatin glycoproteins in potato tubers, patatin hprnai lines showed a normal phenotype during tissue culture and greenhouse cultivation.
furthermore, patatin-knockdown potato tubers allowed rapid purification of other glycoproteins without contamination of patatin.
therefore, patatin-knockdown potato tubers appear to have strong potential as a plant host for the production of human therapeutic glycoproteins.

 the potato is the world's fourth most important crop. potato tuber proteins are categorized into three groups: patatins, protease inhibitors, and other proteins. patatins are a family of glycoproteins and represent up to 40% of the total soluble protein in potato tubers. patatins accumulate in the vacuoles of tubers and leaves and are mainly found in parenchyma cells of potato tubers. two classes of patatin gene families have been identified in potato plants. class i transcripts, which lack the nucleotides in the 5'-untranslated region, were found to be tuber specific, whereas class ii transcripts, which bear the nucleotides, were found in both tubers and roots. patatin genes are strongly expressed in tubers but occur at very low levels in other tissues. patatins are approximately 40– kda in size and are the major storage protein of potato tubers. the roles of patatins are proposed to include fatty acid esterase, lipid acyl hydrolase, and acyltransferase activities. patatins are also known to have antioxidant activities. however, the physiological functions of patatins in potato tubers are not fully understood. potato tubers have been used as a plant host for the production of recombinant human proteins and vaccines. a study of transgenic potato plants expressing a human serum albumin gene revealed that recombinant albumin accumulated up to % of total soluble tuber protein in the plants. the low level of target protein expression is a barrier to recombinant protein production in plants. in addition, the steps involved in purification of target proteins are major cost factors in plant-based protein production. the phenomenon of gene silencing was first observed in the petunia. gene silencing is a eukaryotic genome defense system against viruses and mobile dna elements that works by processing double-stranded rna into short interfering rna. rna interference refers to a multi-step process, including the introduction of double-stranded rna into a cell, cleavage of dsrna into short interfering rna 21– nt in size, formation of rna-induced silencing complex, degradation of complementary mrna, and suppression of target gene expression. rnai technology has become a powerful tool for the study of the functions of individual genes in a range of organisms. hairpin rna, a class of short interfering rna, is composed of two target sequences in an inverted repeat orientation with a spacer between. the two target sequences form a duplex rna with a loop. hprna interference has proven to be an effective tool for the study of the functions of several genes, including the pigment biosynthesis gene chalcone synthase, the ethylene-signaling gene, and the flowering repression gene in arabidopsis. in this study, we utilized the hprnai-based reverse genetic approach to knock down various patatin isoforms in potato tubers. the objective of this study was to provide patatin knockdown potato tubers as a heterologous expression system for making human-like glycoproteins.  construction of a patatin-specific hairpin rnai vector and potato transformation prior to the construction of an hprnai vector for suppression of patatin expression, the sequences of known genes in the patatin multi-gene family in the potato were downloaded from the ncbi database and aligned for a thorough comparison of the genes. great similarities between the patatin genes were found to exist at the nucleotide level. a highly conserved nt fragment of pat3-k, nucleotides 188– was pcr amplified using desiree as template. half of the pcr fragments were inserted into the xhoi – kpni site of pkannibal vector in the sense orientation and the other half of the pcr fragments were inserted into the xbai – clai site in the antisense orientation. the patatin-hprnai frame in pkannibal was confirmed by restriction analysis and nucleotide sequencing. the pkannibal vector was digested with noti enzyme. noti fragments were cloned into part binary vector, resulting in a patatin-specific hprnai vector, which was driven by camv 35s promoter. the patatin hprnai binary vector was transformed into potato plants via agrobacterium-mediated transformation. ten independent patatin-hprnai potato lines were selected on kanamycin. the transgene in the hprnai lines was confirmed by pcr screening with npt ii primers and camv 35s primers. all ten of these lines had the npt ii gene and were resistant to kanamycin. seven of the regenerated kanamycin-resistant potato plants were further analyzed by southern blot to determine the number of t-dna inserts. patatin hprnai lines and each had one t-dna insert. analysis of patatins on 1d sds-page gel to determine the relative efficacy of gene silencing between patatin hprnai lines, one-dimensional sds-page analysis was initially performed using total soluble protein extracted from two month old mature potato tubers of the hprnai and wt plants grown in greenhouse. as expected, the total protein of wt plants showed a thick band corresponding to patatin on the sds-page gel. to our surprise, except for line the patatin hprnai lines did not show patatin bands on the gel. the patatin level in hprnai line was similar to the wt plants. based on the amounts of patatins, gene silencing seemed to be highly effective in the individual lines where the patatin-hprna was expressed. these data indicate that the inverted repeat sequence of partial patatin gene pat3-k effectively induces gene silencing. analysis of patatin variants on 2d page gel based upon the levels of patatin in the 1d gel, we chose four patatin knockdown lines for further analysis of various patatin variants using two-dimensional page gel electrophoresis. our 2d gel analysis of wt and line mature tuber protein confirmed that the presumed multiple patatin spots at kda had disappeared. other patatin hprnai lines showed patterns in patatin reduction similar to line. overall, 2d gel analysis of the patatin-hprnai line revealed that the number and size of patatin spots significantly reduced, whereas other protein spots almost remained unchanged. analysis of pat3-k mrnas in hprnai lines using real-time rt-pcr to confirm the data obtained from 1d sds-page and 2d page gel electrophoresis, a real-time rt-pcr was performed using total rna extracted from mature potato tubers of ten independent patatin-hprnai lines grown in greenhouse. we used a primer pair designed against the most highly conserved sequence in the patatin gene family. the levels of various patatin transcripts present in hprnai lines were measured by a quantitative real-time rt-pcr with the primers. as shown in figure real-time rt-pcr revealed that patatin transcripts in ten patatin-hprnai lines were significantly reduced. patatin hprnai lines and showed significant reductions in patatin transcripts compared to wt plants. these data are consistent with the data obtained from 1d and 2d gel analysis. the expression of the patatin-hprna presumably led to effective inhibition of the expression of the pat3-k gene and other members of the gene family. in vitro- and ex vitro-grown patatin-hprnai lines showed a normal growth pattern and phenotype to examine the effects of gene silencing on the phenotype, tuber number, and average tuber weight of patatin-hprnai potato plants, the transgenic potato plants were cultured in vitro and also grown in a greenhouse. during in vitro culture, the transgenic potato plants showed a normal morphologic characteristic and growth pattern in patatin hprnai lines. ten patatin-hprnai greenhouse-grown potato plants were harvested and the number of tubers per potato plant and average tuber weight were compared with wild-type. the number of tubers in patatin hprnai lines ranged from to, whereas that of wild-type plants was. average tuber weight in patatin hprnai lines and wild-type was variable between the plants. though average tuber weight of patatin hprnai lines seemed to be a bit smaller than that of wild-type, there were no significant differences. overall, patatin-knockdown potato plants showed a normal growth pattern and phenotype during in vitro culture and greenhouse cultivation. patatin-knockdown potatoes reduced additional purification step of glycoproteins patatin-knockdown potatoes are likely to have a few advantages in terms of glycoprotein purification, compared to wild-type potatoes. to confirm the possible advantages of patatin-knockdown potatoes, potato glycoproteins were purified by affinity chromatography on sepharose-coupled concanavalin a and the purified glycoproteins were monitored by running a sds-page gel. the content of total soluble proteins was not reduced in transgenic plants, though patatin was almost completely knocked down. in our purification system, the yields of glycoproteins were %, % and %. the level of glycoproteins in wt was higher than transgenic plants. in addition, sds-page analysis showed that the fraction of purified glycoproteins in wild-type potatoes has two major bands. one was patatin and the other was a band in size 26– kda. in contrast, patatin-rnai lines had only one major band. therefore, no further purification steps to remove patatin were required. discussion patatins are considered to be a major storage protein in potato tubers. here we report that by suppressing the expression of the potato patatin multi-gene family, transgenic potato tubers accumulated dramatically low levels of patatins. this was achieved by rna interference that was first discovered in caenorhabditis elegans. in the present study, we successfully employed rnai technology to produce patatin knockdown/free potato tubers to eventually provide potato tubers as a heterologous expression system for the production of human therapeutic glycoproteins. the potato genome has 64– patatin dna copies, and most of the dna copies do not show much allelic variation based on restriction fragment length polymorphism mapping. a study of patatins present in cv. kuras showed that patatins are 84–96% identified at amino acid sequence levels. when kuras tuber protein spots from coomassie brilliant blue-stained 2d gels were assigned using tryptic pmf analysis, the major acidic protein spots, 40– kda in size, were patatin variants, and more than patatin spots were observed on the 2d gel. all known potato patatins are grouped into a large family of multi-genes sharing 80–95% identity at the nucleotide level and clustered into three clades including pat pat and pat. when we aligned nucleotide sequences of patatin genes from kuras and kennebec, patatins of both cultivars had more than 90% identity. thus, rnai appeared to be a suitable reverse genetic approach for producing patatin-knockdown potato tubers. based upon sequence similarity within potato patatin genes available from ncbi, a partial cdna of pat3-k was chosen as a target sequence to silence patatin multi-genes of desiree. ten independent patatin-hprnai lines were generated. in order to test the effect of gene silencing of patatins, the total soluble protein extracted from patatin hprnai lines was analyzed on 1d sds-page gel. patatin bands were detected in line but not in nine other hprnai lines. since patatins account for ~40% of total soluble protein in potato tubers, the highly reduced patatin content provided an easy measurement of the degree of gene silencing in the individual hprnai lines. the observed suppression of patatins in patatin hprnai lines was analyzed in more detail using two-dimensional gel electrophoresis. 2d gel of wt plants showed typically thick/various patatin spots of to kda, whereas major protein spots were not observed in the gel of hprnai line. based upon the measurement of the relative patatin content between the hprnai line and the wt plant, patatins in line were reduced by about 99%. the results of 2d gel analysis suggested that individual genes of the patatin multi-gene family were significantly knocked down by rna interference. a study of patatin antisense transgenic potato plants showed that the amount of patatin proteins in the plants is maximally reduced by 75–90%. our 2d gel analysis showed that patatin hprnai suppressed up to 99% of potato patatins. the data demonstrated that the rnai approach seems to be much more effective than the antisense rna approach. in addition to the significant reduction of potato patatins, the level of patatin transcripts was examined by a quantitative real-time rt-pcr. the levels of patatin transcripts were dependent on the lines. the maximum reduction of 99% in the transcript level of pat3-k was observed in line in comparison with that of the control pcr. the decreased levels of transcripts in patatin-hprnai line were significantly consistent with the suppression of patatins. since primers of real-time rt-pcr were designed at the highly conserved gene, the dramatic reduction in pat3-k transcripts probably includes the knockdown of other patatin genes as well. furthermore, based upon the fact that various patatin spots were not observed in the 2d gel of patatin hprnai line the transcripts of most patatin genes would be effectively degraded by rna interference. overall, the maximum level of reduction in patatin rnas and proteins in the hprnai lines was approximately 99%. human-like glycoproteins have been produced in plant systems, including potato and tobacco. use of more advantageous protein expression systems is necessary for plant-based human-like glycoprotein production because the yield of recombinant proteins is very low and because expensive purification steps are required. patatin knockdown potatoes could be used as a suitable expression host for the production of human-like glycoproteins, since very low endogenous glycoprotein patatins might make a relatively selective purification of human-like glycoproteins easier, with less patatin contamination. furthermore, the use of patatin tuber specific promoter and n-terminal targeting sequences could easily drive the expression of human glycoproteins in the vacuoles of patatin knockdown potato tubers. glycosylation, the attachment of oligosaccharides to proteins, is one of the major posttranslational modifications of proteins in plants and animals. in most therapeutic glycoproteins, the sugar is attached to the asn residue of a tripeptide asn-x-ser/thr of the protein and/or thr or ser residues in the peptide backbone. the processing of the n-linked glycans occurs in er and golgi apparatus. plant and mammalian n-glycan maturations differ only in the late golgi apparatus. in terms of the production of human therapeutic glycoproteins in plant systems, it is necessary to inhibit undesirable plant-specific posttranslational modifications in plants and to glycosylate target proteins in a manner similar to human systems via genetic engineering. therefore, the first prerequisite for the production of human therapeutic glycoproteins in potato tubers is to suppress the expression of major glycoprotein "patatins". thus, the patatin-knockdown potato tuber would be a practical candidate to host the selective production of human therapeutic glycoproteins.  rna silencing technology was used to produce patatin-knockdown potato tubers. a target sequence of highly conserved gene in the patatin multi-gene family was amplified for the construction of a patatin-specific hairpin rnai vector. the effects of gene silencing in patatin-hprnai lines were analyzed at the protein level by both 1d and 2d page gel electrophoresis. in addition, the gene silencing effects were confirmed at the mrna level by a quantitative real-time rt-pcr. dependent on the patatin hprnai line, almost all of the individual patatin variants were reduced by approximately 99% at both the protein and mrna levels in line despite a significant reduction of patatin glycoproteins in potato tubers, patatin hprnai lines showed a normal phenotype during tissue culture and greenhouse cultivation. furthermore, patatin-knockdown potato tubers allowed rapid purification of other glycoproteins without contamination of patatin. therefore, patatin-knockdown potato tubers appear to have strong potential as a plant host for the production of human therapeutic glycoproteins. 
dependent upon the patatin hprnai line, patatins decreased by approximately 99% at both the protein and mrna levels. during glycoprotein purification, patatin-knockdown potato tubers allowed rapid purification of other potato glycoproteins with less contamination of patatins. more importantly, patatin-knockdown potato tubers appear to be an ideal host for the production of human therapeutic glycoproteins, because they eventually allow fast, easy purification of recombinant proteins, with less contamination from potato glycoprotein patatins. increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. patatin-specific hprnai effectively suppressed the expression of a majority of patatin variants in potato tubers via the specific degradation of individual mrnas of the patatin multi-gene family. the effects of rna interference were characterized at both the protein and mrna levels using 1d and 2d sds/page and quantitative real-time rt-pcr analysis. however, the phenotype of hprnai lines was not distinguishable from wild-type potato plants under both in vitro and ex vitro growth conditions. potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins. patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. in the present study, potato patatins were eliminated almost completely via rna interference technology to develop potato tubers as a more efficient protein expression system. the gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels. based upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence of patatin gene pat3-k in potato plants was amplified for the construction of a patatin-specific hairpin rnai vector.
 patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins. increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. in the present study, potato patatins were eliminated almost completely via rna interference technology to develop potato tubers as a more efficient protein expression system. the gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.  based upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence of patatin gene pat3-k in potato plants was amplified for the construction of a patatin-specific hairpin rnai vector. the camv 35s promoter-driven patatin hprnai vector was transformed into the potato cultivar desiree by agrobacterium-mediated transformation. ten transgenic potato lines bearing patatin hprna were generated. the effects of rna interference were characterized at both the protein and mrna levels using 1d and 2d sds/page and quantitative real-time rt-pcr analysis. dependent upon the patatin hprnai line, patatins decreased by approximately 99% at both the protein and mrna levels. however, the phenotype of hprnai lines was not distinguishable from wild-type potato plants under both in vitro and ex vitro growth conditions. during glycoprotein purification, patatin-knockdown potato tubers allowed rapid purification of other potato glycoproteins with less contamination of patatins.  patatin-specific hprnai effectively suppressed the expression of a majority of patatin variants in potato tubers via the specific degradation of individual mrnas of the patatin multi-gene family. more importantly, patatin-knockdown potato tubers appear to be an ideal host for the production of human therapeutic glycoproteins, because they eventually allow fast, easy purification of recombinant proteins, with less contamination from potato glycoprotein patatins. 