Development of gene expression system in a marine diatom using viral promoters of a wide variety of origin

Abstract

Promoter sequences of the cytomegalovirus (PCMV), the rous sarcoma virus long terminal repeat (PRSV-LTR) and the cauliflower mosaic virus 35s (PCaMV35s) were ligated with the beta-glucuronidase (GUS) gene, uidA, and were introduced into cells of the marine diatom, Phaeodactylum tricornutum. Transformants were selected on a 100 mg l(-1) Zeocin plate, and Zeocin-resistant clones were further selected by the occurrence of GUS activity. Two to 10 GUS-positive clones were obtained, and GUS activities in these transformants did not change in response to changes in ambient CO(2) concentration except that the PRSV-LTR was weakly activated in air. These results indicate that a wide spectrum of viral promoters originating from mammalian, avian and plant hosts can operate as constitutive promoters in a marine diatom. The CO(2) responsive promoter sequence of the chloroplastic carbonic anhydrase gene in P. tricornutum (Pptca1) with a deleted initiator region was ligated with the minimal region of the PCMV followed by uidA and was introduced into P. tricornutum. GUS expression in the resulting transformants was clearly regulated by CO(2), that is, GUS expression was stimulated in air to about 10-fold than that in cells grown in 5% CO(2). However, the CO(2) response disappeared when the core regulatory region of Pptca1 (-76 to -11 bp) was removed. The regulative function of the endogenous diatom promoter was thus maintained after fusion with an extrinsic viral promoter. These results indicate that diatom cells accommodate a wide range of transcriptional system from beyond the plant kingdom and that an efficient transcriptional system could potentially be constructed in marine diatoms by selecting an appropriate set of viral promoter and functional cis elements.

Rapid induction of GFP expression by the nitrate reductase promoter in the diatomPhaeodactylum tricornutum

“…Although there is some information available regarding the functionality of heterologous regulatory DNA sequences or promoters in diatoms (Brakemann et al, 2008; Dunahay, Jarvis & Roessler, 1995; Falciatore et al, 1999; Kadono et al, 2015; Sakaue, Harada & Matsuda, 2008), all the widely utilised transformation systems for the diatoms Phaeodactylum tricornutum (Falciatore et al, 1999; Zaslavskaia et al, 2000), Cylindrotheca fusiformis (Fischer et al, 1999) and Thalassiosira pseudonana (Poulsen, Chesley & Kröger, 2006) employ promoters derived from the respective target organism. The availability of genome sequence data for several diatoms (Armbrust et al, 2004; Bowler et al, 2008; Lommer et al, 2012) allows the identification of putative promoter sequences that can be tested regarding their usability.…”

Light and CO2/cAMP Signal Cross Talk on the Promoter Elements of Chloroplastic β-Carbonic Anhydrase Genes in the Marine Diatom Phaeodactylum tricornutum

“…Our previous studies have demonstrated that the endogenous ptca1 and ptca2 showed, respectively, about 25 and 3.5 times increase in their transcript levels by acclimating from 5% CO 2 to air Harada et al, 2006) and the reporter construct of Pptca1 fused with the b-glucuronidase (GUS) gene, uidA, was a quantitative indicator for the CO 2 response of Pptca1 in transformant cells expressing this fusion reporter construct (Harada et al, , 2006Sakaue et al, 2008, Ohno et al, 2012. We thus used this GUS reporter system to identify the critical element required for switching Pptcas activity in response to light/CO 2 by means of sequence manipulations of heterologously introduced promoter constructs.…”

Microalgae, Functional Genomics and Biotechnology

“…The same strategy, using HSP70 fused to CAb (chlorophyll-binding protein), was reported in the charophyte Closterium peracerosum (Abe et al, 2011). Usual plant promoters, such as the cauliflower mosaic virus 35S with Ubiquitin-Ω, have been also tested in some microalgae (Kumar et al, 2004, Jarvis and Brown, 1991, Chen et al, 2001, Wang et al, 2007, and recently the 35S promoter demonstrated efficiency in the diatom P. tricornutum (Sakaue et al, 2008), Chlorophytes Haematococcus sp. (Kathiresan et al, 2009) and Dunaliella bardawil (Anila et al, 2011) and the Heterokonta Nannochloropsis sp.…”