Kim Yoon-Sik, Lee Yong-Hwa, Kim Hyun-Soon, Kim Mi-Sun, Hahn Kyu-Woong, Ko Jeong-Heon, Joung Hyouk, Jeon Jae-Heung
Plant Genome Research Center, KRIBB, Daejeon 305-806, Korea.
BMC Biotechnol. 2008 Apr 3;8:36. doi: 10.1186/1472-6750-8-36.
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.
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.
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.
由多基因家族编码的patatins是马铃薯块茎中主要的贮藏糖蛋白之一。最近，马铃薯块茎已成为生产人类治疗性糖蛋白（疫苗）的生物反应器。提高重组蛋白产量、将产生的蛋白靶向特定细胞区室以及减少昂贵的蛋白纯化步骤是植物生物技术中的重要研究目标。在本研究中，通过RNA干扰（RNAi）技术几乎完全消除了马铃薯patatins，以将马铃薯块茎开发为更高效的蛋白表达系统。在个体同工型水平上检测了转基因马铃薯植株中patatins的基因沉默效果。
基于patatins多基因家族内的序列相似性，扩增了马铃薯（Solanum tuberosum L.）中patatin基因pat3-k1 [GenBank登录号：DQ114421]的高度保守靶序列（635个核苷酸），用于构建patatin特异性发夹RNAi（hpRNAi）载体。通过农杆菌介导的转化将CaMV 35S启动子驱动的patatin hpRNAi载体转化到马铃薯品种Desiree中。产生了10个携带patatin hpRNA的转基因马铃薯株系。使用一维和二维SDS/PAGE以及定量实时RT-PCR分析在蛋白质和mRNA水平上表征RNA干扰的效果。取决于patatin hpRNAi株系，patatins在蛋白质和mRNA水平上均下降了约99%。然而，在体外和离体生长条件下，hpRNAi株系的表型（如马铃薯块茎的数量和大小、平均块茎重量、生长模式等）与野生型马铃薯植株没有区别。在糖蛋白纯化过程中，敲除patatin的马铃薯块茎能够快速纯化其他马铃薯糖蛋白，且patatins的污染较少。
patatin特异性hpRNAi通过特异性降解patatin多基因家族的各个mRNA，有效地抑制了马铃薯块茎中大多数patatin变体的表达。更重要的是，敲除patatin的马铃薯块茎似乎是生产人类治疗性糖蛋白的理想宿主，因为它们最终能够快速、轻松地纯化重组蛋白，且马铃薯糖蛋白patatins的污染较少。
