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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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Related in: MedlinePlus

Characterization of T-DNA insertions in patatin hpRNAi lines. (A) PCR amplification of npt II gene in ten patatin hpRNAi lines. The PCR results confirmed that lanes 1 to 10 are patatin hpRNAi lines. Lane PC (DNA of hpRNAi vector was used as a template for PCR) or lane WT (genomic DNA of WT plants was used as a template for PCR) is the positive or negative control, respectively. Lanes 1 to 10 are patatin hpRNAi potato lines. (B) DNA gel blot of wild-type and seven patatin hpRNAi lines. Genomic DNA was digested with XbaI and probed with npt II gene probe. Lines 4, 7, 8, and 10 each have one T-DNA insertion.
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Figure 2: Characterization of T-DNA insertions in patatin hpRNAi lines. (A) PCR amplification of npt II gene in ten patatin hpRNAi lines. The PCR results confirmed that lanes 1 to 10 are patatin hpRNAi lines. Lane PC (DNA of hpRNAi vector was used as a template for PCR) or lane WT (genomic DNA of WT plants was used as a template for PCR) is the positive or negative control, respectively. Lanes 1 to 10 are patatin hpRNAi potato lines. (B) DNA gel blot of wild-type and seven patatin hpRNAi lines. Genomic DNA was digested with XbaI and probed with npt II gene probe. Lines 4, 7, 8, and 10 each have one T-DNA insertion.

Mentions: Prior to the construction of an hpRNAi vector for suppression of patatin expression, the sequences of 29 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 (> 90%) between the patatin genes were found to exist at the nucleotide level (data not shown). A highly conserved 635 nt fragment of pat3-k1 (GenBank: DQ114421), nucleotides 188–798, 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 pART27 binary vector, resulting in a patatin-specific hpRNAi vector, which was driven by CaMV 35S promoter (Figure 1). The patatin hpRNAi binary vector was transformed into potato plants (cv. Desiree) 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 (Figure 2A) and CaMV 35S primers (data not shown). 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 (Figure 2B). Patatin hpRNAi lines 4, 7, 8, and 10 each had one T-DNA insert.


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)

Characterization of T-DNA insertions in patatin hpRNAi lines. (A) PCR amplification of npt II gene in ten patatin hpRNAi lines. The PCR results confirmed that lanes 1 to 10 are patatin hpRNAi lines. Lane PC (DNA of hpRNAi vector was used as a template for PCR) or lane WT (genomic DNA of WT plants was used as a template for PCR) is the positive or negative control, respectively. Lanes 1 to 10 are patatin hpRNAi potato lines. (B) DNA gel blot of wild-type and seven patatin hpRNAi lines. Genomic DNA was digested with XbaI and probed with npt II gene probe. Lines 4, 7, 8, and 10 each have one T-DNA insertion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Characterization of T-DNA insertions in patatin hpRNAi lines. (A) PCR amplification of npt II gene in ten patatin hpRNAi lines. The PCR results confirmed that lanes 1 to 10 are patatin hpRNAi lines. Lane PC (DNA of hpRNAi vector was used as a template for PCR) or lane WT (genomic DNA of WT plants was used as a template for PCR) is the positive or negative control, respectively. Lanes 1 to 10 are patatin hpRNAi potato lines. (B) DNA gel blot of wild-type and seven patatin hpRNAi lines. Genomic DNA was digested with XbaI and probed with npt II gene probe. Lines 4, 7, 8, and 10 each have one T-DNA insertion.
Mentions: Prior to the construction of an hpRNAi vector for suppression of patatin expression, the sequences of 29 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 (> 90%) between the patatin genes were found to exist at the nucleotide level (data not shown). A highly conserved 635 nt fragment of pat3-k1 (GenBank: DQ114421), nucleotides 188–798, 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 pART27 binary vector, resulting in a patatin-specific hpRNAi vector, which was driven by CaMV 35S promoter (Figure 1). The patatin hpRNAi binary vector was transformed into potato plants (cv. Desiree) 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 (Figure 2A) and CaMV 35S primers (data not shown). 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 (Figure 2B). Patatin hpRNAi lines 4, 7, 8, and 10 each had one T-DNA insert.

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