Mallory Allison C, Parks Graham, Endres Matthew W, Baulcombe David, Bowman Lewis H, Pruss Gail J, Vance Vicki B
University of South Carolina, Columbia, SC 29208, USA.
Nat Biotechnol. 2002 Jun;20(6):622-5. doi: 10.1038/nbt0602-622.
Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) "amplicon" lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS). In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction. Even though the amplicon locus is silenced, the level of beta-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to approximately 3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.
许多生物技术应用都需要在植物中实现转基因的高水平表达。实现这一目标的一种策略是构建马铃薯X病毒（PVX）“扩增子”株系：即编码携带目标基因的复制型RNA病毒载体的转基因株系。其设想是扩增子转基因的转录会启动病毒RNA复制和基因表达，从而产生非常高水平的目标基因产物。然而，这种方法失败了，因为在烟草和拟南芥中，每个扩增子转基因都受到转录后基因沉默（PTGS）的影响。在PTGS中，转基因虽能转录，但转录本由于序列特异性靶向和破坏而无法积累。尽管扩增子位点被沉默，但PVX/GUS株系中β-葡萄糖醛酸酶（GUS）的活性水平与一些从常规（未沉默）GUS位点表达GUS的转基因株系相似。这一结果表明，如果能够克服PTGS，并且由此产生的植物不患严重的病毒病，那么最初设想的扩增子的高水平表达是可以实现的。在此我们报告，通过将扩增子方法与使用PTGS的病毒抑制因子烟草蚀纹病毒（TEV）辅助成分蛋白酶（HC-Pro）相结合，可以实现转基因的高水平表达。共表达HC-Pro和PVX/GUS扩增子的成熟烟草植株的叶片中，GUS积累量约占总蛋白的3%。此外，高水平表达不会出现病毒症状，并且当HC-Pro由突变转基因表达时，也不会出现有害的发育表型。
