A new molecular tool for transgenic diatoms -: Control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette
A new molecular tool for transgenic diatoms -: Control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette
复制标题
DOI：
10.1111/j.1742-4658.2005.04760.x
复制
发表时间：
2005-07-01
期刊：
影响因子：
5.4
通讯作者：
Kröger, N
中科院分区：
文献类型：
--
作者：
Poulsen, N;Kröger, N
自近期完成的假微型海链藻基因组计划以来，硅藻生物学研究进入了后基因组时代。然而，可用于硅藻基因操作的分子工具仍然很少，阻碍了硅藻基因在体内的功能分析。在这里，我们描述了转基因硅藻中诱导基因表达的第一种方法。该方法使用含有源自硅藻 Cylindrotheca fusiformis 硝酸盐还原酶基因的启动子 (Pnr) 和终止子 (Tnr) 元件的 DNA 盒。通过使用绿色荧光蛋白 (gfp) cDNA 作为报告基因，证明当细胞在铵离子存在下生长时，Pnr/Tnr 盒控制下的基因表达被关闭，而当细胞转移到含有硝酸盐的培养基时，在 4 小时内打开。在无氮培养基中孵育细胞会开启 gfp 基因的转录，但 gfp mRNA 不会翻译成蛋白质。通过添加硝酸盐可以释放这种翻译阻断，导致 GFP 快速产生，延迟时间大幅缩短，仅为 1 小时。总之，我们已经证明，Pnr/Tnr 盒能够诱导基因表达，并控制转基因硅藻中 mRNA 和蛋白质表达的水平和时间。
Research in diatom biology has entered the postgenomic era since the recent completion of the Thalassiosira pseudonana genome project. However, the molecular tools available for genetic manipulation of diatoms are still sparse, impeding the functional analysis of diatom genes in vivo. Here we describe the first method for inducible gene expression in transgenic diatoms. This method uses a DNA cassette containing both promoter (Pnr) and terminator (Tnr) elements derived from the nitrate reductase gene of the diatom Cylindrotheca fusiformis. By using green fluorescent protein (gfp) cDNA as a reporter gene, it is demonstrated that gene expression under the control of the Pnr/Tnr cassette is switched off when cells are grown in the presence of ammonium ions and becomes switched on within 4 h when cells are transferred to medium containing nitrate. Incubating cells in nitrogen-free medium switches on transcription of the gfp gene, yet gfp mRNA does not become translated into protein. This block on translation is released by the addition of nitrate, resulting in rapid onset of GFP production with a drastically reduced delay time of only 1 h. Altogether we have demonstrated that the Pnr/Tnr cassette enables inducible gene expression and control of both the level and timing of mRNA and protein expression in transgenic diatoms.