Magee Alan M, MacLean Daniel, Gray John C, Kavanagh Tony A
Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland.
Transgenic Res. 2007 Aug;16(4):415-28. doi: 10.1007/s11248-006-9045-z. Epub 2006 Nov 14.
Transcription of plastid transgenes by plastid-targeted T7 RNA polymerase (ptT7RNAP) during early seedling development in tobacco was associated with a pale-green leaf phenotype, depletion of plastid rRNAs and arrest of shoot development. Extensive analysis of mutant seedlings at the transcript level using DNA microarrays and RNA gel blotting revealed severe disruption of plastid rRNA accumulation at 4-days post-germination and reduced transcript accumulation for the essential gene clpP. Several nuclear genes encoding plastid proteins were differentially regulated in mutant seedlings over time. Ef-Tu was upregulated at 4-days post-germination and then subsequently downregulated, while RbcS was already downregulated at this early time point. The downregulation of nuclear genes encoding plastid proteins suggests disruption of plastid-to-nucleus signalling. In contrast, transcripts of three plastid genes showed increased accumulation in mutant seedlings. Transcripts of ndhC and ndhK accumulated at high levels possibly due to T7RNAP-mediated enhancement of transcription, while ptT7RNAP-mediated transcription through the phage T7 Tphi terminator into the adjacent plastome increased the level of accD transcripts. The leakiness of the Tphi terminator has implications for the use of T7RNAP-based expression systems in plastid biotechnology.
在烟草幼苗早期发育过程中，质体靶向的T7 RNA聚合酶（ptT7RNAP）对质体转基因的转录与浅绿色叶片表型、质体rRNA的消耗以及茎发育的停滞有关。使用DNA微阵列和RNA凝胶印迹在转录水平对突变幼苗进行的广泛分析显示，发芽后4天时质体rRNA积累严重中断，必需基因clpP的转录本积累减少。随着时间的推移，几个编码质体蛋白的核基因在突变幼苗中受到不同程度的调控。发芽后4天时，延伸因子Tu（Ef-Tu）上调，随后下调，而核酮糖-1,5-二磷酸羧化酶小亚基（RbcS）在这个早期时间点就已经下调。编码质体蛋白的核基因的下调表明质体到细胞核的信号传导受到破坏。相比之下，三个质体基因的转录本在突变幼苗中积累增加。ndhC和ndhK的转录本高水平积累可能是由于T7RNAP介导的转录增强，而ptT7RNAP通过噬菌体T7 Tphi终止子介导的转录进入相邻的质体基因组增加了accD转录本的水平。Tphi终止子的渗漏对质体生物技术中基于T7RNAP的表达系统的使用具有重要意义。
