Chu Lili, Ewe Daniela, Río Bártulos Carolina, Kroth Peter G, Gruber Ansgar
Fachbereich Biologie, Universität Konstanz , Konstanz , Germany.
Fachbereich Biologie, Universität Konstanz, Konstanz, Germany; Current affiliation: Centre Algatech, Institute of Microbiology, The Czech Academy of Science, Třeboň, Czech Republic.
PeerJ. 2016 Aug 25;4:e2344. doi: 10.7717/peerj.2344. eCollection 2016.
An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns.
可控转基因表达的一个基本前提是选择合适的启动子。在硅藻三角褐指藻模型中，转基因表达最常用的启动子是光依赖型lhcf1启动子（源自两个编码岩藻黄素叶绿素a/c结合蛋白的内源基因）和硝酸盐依赖型nr启动子（源自内源硝酸还原酶基因）。在本研究中，我们通过流式细胞术、显微镜检查和蛋白质印迹法，在培养基中通过改变氮源诱导表达后，研究了独立遗传转化的三角褐指藻细胞系中，硝酸还原酶启动子控制下绿色荧光蛋白（GFP）报告基因的时间依赖性表达。在所有研究的细胞系中，培养基更换后1小时GFP荧光开始增加，在2至3小时之间观察到最快的增加速率。即使将细胞转移到铵培养基中，荧光在长达7小时内仍继续略有增加。随后GFP荧光的下降比增加慢得多，这可能是由于GFP的稳定性。对几个用基于nr的构建体转化的细胞系的研究表明，即使在没有硝酸盐的情况下，启动子也可能表现出残余活性。此外，我们观察到独立细胞系之间基因表达存在很大差异，这强调了全面表征转基因细胞系及其个体表达模式的重要性。
