Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany.
Curr Genet. 2010 Aug;56(4):383-9. doi: 10.1007/s00294-010-0304-4. Epub 2010 May 7.
Several RNA silencing strategies employing antisense or inverted repeat constructs have been applied to Chlamydomonas reinhardtii. Problems inherent to these strategies, like off-target effects by unpredictable generation of siRNAs, were solved previously by constructs allowing for routine expression of specific artificial microRNAs (amiRNAs). Yet missing was a routine tool for inducible amiRNA expression, which to establish was the aim of this work. For this, we equipped a recently developed amiRNA expression vector with the NIT1 promoter, which is repressed by ammonium and activated by nitrate. We tested this conditional amiRNA vector with heat shock factor 1 (HSF1) as target. HSF1 transcripts in transformants were already reduced ~2 h after transfer from ammonium to nitrate-containing medium. In contrast, HSF1 protein levels declined only ~8 h after the shift and were strongly reduced after 24 h, suggesting that HSF1 is a stable protein and diluted out by growth. HSF1 levels recovered partly when transformant cells were shifted back to ammonium for 72 h. Transformants developed thermosensitivity only on nitrate and thermosensitivity correlated with strong reduction in HSF1 levels, hence supporting our earlier conclusion that HSF1 is a key regulator for thermotolerance in Chlamydomonas.
几种 RNA 沉默策略采用反义或反向重复构建体已应用于莱茵衣藻。这些策略固有的问题，如通过不可预测的 siRNA 产生产生的脱靶效应，以前通过允许常规表达特定人工 microRNA (amiRNA) 的构建体得到解决。然而，缺少常规的诱导型 amiRNA 表达工具，这是这项工作的目的。为此，我们用受铵抑制、硝态氮激活的 NIT1 启动子装备了最近开发的 amiRNA 表达载体。我们用热休克因子 1 (HSF1) 作为靶标测试了这种条件 amiRNA 载体。在从铵盐转移到含硝酸盐的培养基后约 2 小时，转化体中的 HSF1 转录本已被降低。相比之下，HSF1 蛋白水平在转变后仅约 8 小时下降，24 小时后强烈下降，表明 HSF1 是一种稳定的蛋白质，并通过生长稀释。当转化细胞在 72 小时内转回铵盐时，HSF1 水平部分恢复。转化体仅在硝酸盐上表现出热敏性，热敏性与 HSF1 水平的强烈降低相关，因此支持我们之前的结论，即 HSF1 是莱茵衣藻耐热性的关键调节剂。
