Cloning of a novel endogenous promoter for foreign gene expression in Phaeodactylum tricornutum

Abstract

Phaeodactylum tricornutum is a model diatom, and its genomic sequence data and expressed sequence tag databases are available. This study was to discover a new endogenous promoter that drives strong constitutive expression of a protein of interest in P. tricornutum. To find promoter candidates, the intracellular proteins of P. tricornutum grown to stationary phase were extracted and identified by LC？MS/MS. Glutamine synthetase (GLNA) was one of the most abundantly expressed proteins during the stationary phase. Promoter is usually located on 5′ upstream region of open reading frame of the gene. Thus, two fragments of 5′ upstream region of the GLNA gene as putative promoters, 501 and 997？bp long, were amplified and cloned into enhanced Green Fluorescent Protein (eGFP) reporter systems. The constructed reporter systems were transformed into P. tricornutum and the eGFP expression levels were compared to those of reporter systems using the promoters of fcpA (fucoxanthin chlorophyll a/c binding protein A) and CIP1 (putative replication-associated proteins of a Chaetoceros lorenzianus-infecting DNA virus) as controls. The expression of eGFP driven by either GLNA promoter (501 and 997？bp) was linearly related to cell density, and eGFP was expressed constitutively regardless of the cultivation phase. The eGFP expression level driven by the GLNA promoters was at least 4 times higher than the fcpA-driven eGFP expression level at the stationary phase. The 501 and 997？bp regions of the GLNA promoter had similar activity patterns for transcribing the downstream gene. These results indicate that at least 501-bp region of the GLNA promoter can be used as a strong constitutive promoter in genetic engineering of P. tricornutum. ？ 2016, The Korean Society for Applied Biological Chemistry.