Tungsuchat Tarinee, Kuroda Hiroshi, Narangajavana Jarunya, Maliga Pal
Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ 08854-8020, USA.
Plant Mol Biol. 2006 Jul;61(4-5):711-8. doi: 10.1007/s11103-006-0044-5.
We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.
我们开发了一种用于质体基因激活的新型系统，该系统利用CRE/loxP位点特异性重组系统，通过切除一个阻断序列来创建一个可翻译的阅读框。为了测试该系统，我们将一个无活性的gfp基因导入烟草质体基因组中，位于可选的壮观霉素抗性（aadA）标记基因的下游。aadA基因是阻断序列，其两侧是同向的loxP位点，以便由CRE进行切除。在未激活状态下，gfp从aadA启动子转录，但由于缺乏AUG翻译起始密码子，mRNA无法翻译。通过切除aadA编码片段，将gfp编码区与aadA的翻译起始密码子连接起来，从而激活绿色荧光蛋白（GFP）的表达。携带无活性gfp基因的烟草植株不含可检测水平的GFP。然而，gfp*的激活导致了GFP的积累，证明了CRE诱导的蛋白表达在烟草叶绿体中的实用性。这里描述的基因激活系统将有助于探究质体基因功能，并用于在叶绿体中生产重组蛋白。
