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Development of patatin knockdown potato tubers using RNA interference (RNAi) technology, for the production of human-therapeutic glycoproteins.

Kim YS, Lee YH, Kim HS, Kim MS, Hahn KW, Ko JH, Joung H, Jeon JH - BMC Biotechnol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Genome Research Center, KRIBB, Daejeon 305-806, Korea. yoon1920@kribb.re.kr

ABSTRACT

Background: 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 (vaccines). 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 (RNAi) 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.

Results: Based upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence (635 nts) of patatin gene pat3-k1 [GenBank accession no. DQ114421] in potato plants (Solanum tuberosum L.) was amplified for the construction of a patatin-specific hairpin RNAi (hpRNAi) 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 (e.g. the number and size of potato tuber, average tuber weight, growth pattern, etc.) 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.

Conclusion: 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.

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Real-time RT-PCR of patatin hpRNAi lines. Real-time RT-PCR was performed on RNA extracted from 10 independent hpRNAi lines using primers designed against the patatin gene (pat3-k1). WT is a wild-type positive control. Lanes 1 to 10 are ten independent patatin hpRNAi lines. 18S rRNA was amplified as a control for equal amounts of RNA templates. Real-time RT-PCR results show relative fold changes of pat3-k1 transcript levels in hpRNAi lines in comparison with WT. The error bars indicate mean ± SD. Mean and SD were derived by two independent experiments. pat3-k1 transcript levels were significantly reduced in most hpRNAi lines, compared to the WT control.
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Figure 4: Real-time RT-PCR of patatin hpRNAi lines. Real-time RT-PCR was performed on RNA extracted from 10 independent hpRNAi lines using primers designed against the patatin gene (pat3-k1). WT is a wild-type positive control. Lanes 1 to 10 are ten independent patatin hpRNAi lines. 18S rRNA was amplified as a control for equal amounts of RNA templates. Real-time RT-PCR results show relative fold changes of pat3-k1 transcript levels in hpRNAi lines in comparison with WT. The error bars indicate mean ± SD. Mean and SD were derived by two independent experiments. pat3-k1 transcript levels were significantly reduced in most hpRNAi lines, compared to the WT control.

Mentions: 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 (two month old) 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 4, real-time RT-PCR revealed that patatin transcripts in ten patatin-hpRNAi lines were significantly reduced. Patatin hpRNAi lines 4, 5, 7, 8 and 10 showed significant reductions (95 to 99%) 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-k1 gene and other members of the gene family.


Development of patatin knockdown potato tubers using RNA interference (RNAi) technology, for the production of human-therapeutic glycoproteins.

Kim YS, Lee YH, Kim HS, Kim MS, Hahn KW, Ko JH, Joung H, Jeon JH - BMC Biotechnol. (2008)

Real-time RT-PCR of patatin hpRNAi lines. Real-time RT-PCR was performed on RNA extracted from 10 independent hpRNAi lines using primers designed against the patatin gene (pat3-k1). WT is a wild-type positive control. Lanes 1 to 10 are ten independent patatin hpRNAi lines. 18S rRNA was amplified as a control for equal amounts of RNA templates. Real-time RT-PCR results show relative fold changes of pat3-k1 transcript levels in hpRNAi lines in comparison with WT. The error bars indicate mean ± SD. Mean and SD were derived by two independent experiments. pat3-k1 transcript levels were significantly reduced in most hpRNAi lines, compared to the WT control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335101&req=5

Figure 4: Real-time RT-PCR of patatin hpRNAi lines. Real-time RT-PCR was performed on RNA extracted from 10 independent hpRNAi lines using primers designed against the patatin gene (pat3-k1). WT is a wild-type positive control. Lanes 1 to 10 are ten independent patatin hpRNAi lines. 18S rRNA was amplified as a control for equal amounts of RNA templates. Real-time RT-PCR results show relative fold changes of pat3-k1 transcript levels in hpRNAi lines in comparison with WT. The error bars indicate mean ± SD. Mean and SD were derived by two independent experiments. pat3-k1 transcript levels were significantly reduced in most hpRNAi lines, compared to the WT control.
Mentions: 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 (two month old) 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 4, real-time RT-PCR revealed that patatin transcripts in ten patatin-hpRNAi lines were significantly reduced. Patatin hpRNAi lines 4, 5, 7, 8 and 10 showed significant reductions (95 to 99%) 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-k1 gene and other members of the gene family.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Genome Research Center, KRIBB, Daejeon 305-806, Korea. yoon1920@kribb.re.kr

ABSTRACT

Background: 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 (vaccines). 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 (RNAi) 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.

Results: Based upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence (635 nts) of patatin gene pat3-k1 [GenBank accession no. DQ114421] in potato plants (Solanum tuberosum L.) was amplified for the construction of a patatin-specific hairpin RNAi (hpRNAi) 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 (e.g. the number and size of potato tuber, average tuber weight, growth pattern, etc.) 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus