Siaut Magali, Heijde Marc, Mangogna Manuela, Montsant Anton, Coesel Sacha, Allen Andrew, Manfredonia Alessandro, Falciatore Angela, Bowler Chris
Laboratory of Cell Signalling, Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy.
Gene. 2007 Dec 30;406(1-2):23-35. doi: 10.1016/j.gene.2007.05.022. Epub 2007 Jun 12.
Research into diatom biology has now entered the post-genomics era, following the recent completion of the Thalassiosira pseudonana and Phaeodactylum tricornutum whole genome sequences and the establishment of Expressed Sequence Tag (EST) databases. The thorough exploitation of these resources will require the development of molecular tools to analyze and modulate the function of diatom genes in vivo. Towards this objective, we report here the identification of several reference genes that can be used as internal standards for gene expression studies by quantitative real-time PCR (qRT-PCR) in P. tricornutum cells grown over a diel cycle. In addition, we describe a series of diatom expression vectors based on Invitrogen Gateway technology for high-throughput protein tagging and overexpression studies in P. tricornutum. We demonstrate the utility of the diatom Destination vectors for determining the subcellular localization of a protein of interest and for immunodetection. The availability of these new resources significantly enriches the molecular toolbox for P. tricornutum and provides the diatom research community with well defined high-throughput methods for the analysis of diatom genes and proteins in vivo.
随着三角褐指藻（Phaeodactylum tricornutum）和假微型海链藻（Thalassiosira pseudonana）全基因组序列的近期完成以及表达序列标签（EST）数据库的建立，硅藻生物学研究现已进入后基因组时代。要充分利用这些资源，就需要开发分子工具来在体内分析和调节硅藻基因的功能。为实现这一目标，我们在此报告鉴定了几个可作为内参基因的基因，这些基因可用于在昼夜周期中生长的三角褐指藻细胞中通过定量实时PCR（qRT-PCR）进行基因表达研究。此外，我们描述了一系列基于Invitrogen Gateway技术的硅藻表达载体，用于三角褐指藻中的高通量蛋白质标签和过表达研究。我们展示了硅藻目的载体在确定感兴趣蛋白质的亚细胞定位和免疫检测方面的实用性。这些新资源的可用性显著丰富了三角褐指藻的分子工具库，并为硅藻研究界提供了明确的高通量方法，用于在体内分析硅藻基因和蛋白质。
