Abstract
BackgroundPatatins 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.ResultsBased 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.ConclusionPatatin-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.
Highlights
Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers
NotI fragments were cloned into pART27 binary vector, resulting in a patatin-specific hairpin RNAi (hpRNAi) vector, which was driven by CaMV 35S promoter (Figure 1)
The patatin hpRNAi binary vector was transformed into potato plants
Summary
Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels. Potato tuber proteins are categorized into three groups: patatins, protease inhibitors, and other proteins [1]. Patatins are a family of glycoproteins and represent up to 40% of the total soluble protein in potato tubers [2]. Patatins accumulate in the vacuoles of tubers and leaves and are mainly found in parenchyma cells of potato tubers [3]. Two classes of patatin gene families have been identified in potato plants [4]. Patatin genes are strongly expressed in tubers but occur at very low levels in other tissues [2]
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