Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum.
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.
Laboratory of Cell Signalling, Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy.
Magali
M
Siaut
Marc
M
Heijde
Manuela
M
Mangogna
Anton
A
Montsant
Sacha
S
Coesel
Andrew
A
Allen
Alessandro
A
Manfredonia
Angela
A
Falciatore
Chris
C
Bowler
D000818:Animals; D017377:Diatoms / Q000235:genetics*; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods*; D005822:Genetic Vectors; D016678:Genome; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction