An RNAi system in Physcomitrella patens with an internal marker for silencing allows for rapid identification of loss of function phenotypes
An RNAi system in Physcomitrella patens with an internal marker for silencing allows for rapid identification of loss of function phenotypes
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DOI：
10.1055/s-2005-837597
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发表时间：
2005-05-01
期刊：
影响因子：
3.9
通讯作者：
Quatrano, RS
中科院分区：
文献类型：
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作者：
Bezanilla, M;Perroud, PF;Quatrano, RS
RNAi 是产生功能缺失突变体的强大方法，特别是针对属于大基因家族的基因。我们最近证明 RNAi 在 Physcomitrella patens 苔藓中发挥作用。我们获得了稳定的细胞系，显示核定位 GFP:GUS 融合蛋白 (NLS:GFP:GUS) 的组成型沉默。然而，显示蛋白质沉默的品系不一定具有降低的转录物水平。因此，我们开发了一种系统，可以在沉默感兴趣的基因的同时沉默 NLS:GFP:GUS 报告基因构建体。 Gateway (R) (Invitrogen) 重组盒被整合到这些载体中，以促进许多不同 cDNA 序列的克隆。此外，还生成了包含苔藓基因组 DNA 序列信息的载体，以增加稳定苔藓品系的产量。使用这些构建体进行转化会在 24 小时内产生强烈的沉默，并且在转化后至少一个月内保持稳定。 FtsZ2-1 的功能丧失表型已知，被纳入作为分析表型的测试用例。 100% 沉默 GFP 的再生菌落表现出功能丧失的 FtsZ2-1 表型，验证了使用该系统来分析功能未知的植物基因的表型。
RNAi is a powerful method for generating loss of function mutants, especially for targeting genes belonging to large gene families. We have recently shown that RNAi functions in the moss Physcomitrella patens. We obtained stable lines that show constitutive silencing of a nuclearly localized GFP:GUS fusion protein (NLS:GFP:GUS). However, lines that display silencing of the protein do not necessarily have reduced transcript levels. Therefore, a system has been developed that silences the NLS:GFP:GUS reporter construct at the same time as it silences a gene of interest. Gateway (R) (Invitrogen) recombination cassettes were incorporated into these vectors to facilitate cloning of many different cDNA sequences. In addition, vectors were generated that contain genomic moss DNA sequence information to increase the production of stable moss lines. Transformation with these constructs results in strong silencing within 24 h and is stable for at least a month after transformation. FtsZ2-1, whose loss of function phenotype is known, was incorporated as a test case for analyzing phenotypes. One hundred per cent of regenerating colonies that have silenced GFP exhibit a loss of function FtsZ2-1 phenotype, validating the use of this system to assay phenotypes for plant genes of unknown function.