Name: Keizo Nagasaki

Email: nagasaki@affrc.go.jp

Author: Arisa Miyagawa1, Yuji Tomaru2, Keizo Nagasaki2*, Haruo Yamaguchi3, Masao Adachi3

Author affiliation: 1) Kochi System Glycobiology Center, Kochi University, Kochi, Japan; 2) National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Hiroshima, Japan; 3) Laboratory of Aquatic Environmental Science, Faculty of Agriculture, Kochi University, Kochi, Japan.

Abstract title: Development of marine diatom transformation system using viral promoter

Absstract:

Genetic transformation techniques of marine diatom have been established in only a few species. Several endogenous diatom promoters (ex. fucoxanthin chlorophyll a/c-binding protein gene [fcp] promoter) were used to express extrinsic genes in diatom cells. However, applicable range of endogenous diatom promoters is limited to a few diatom species including a species from which the promoter was derived. The present investigation attempted to develop a transformation system applicable to a wide range of diatom species by using promoters of a diatom-infecting virus. The upstream region (nt -1 to -477) of the putative replication-associated protein gene (CdP1) of CdebDNAV (Chaetoceros debilis DNA virus) (Tomaru et al. 2008) was amplified by PCR. Two plasmids, "pCdP1/nat/TpfcpTer" and "pCdP1/ble/CffcpTer1" were constructed using the Gateway cloning system (Invitrogen Inc.) for transformation of centric and pennate diatoms, respectively; the former plasmid contained nourseothricin resistance gene (nat) with the CdP1 promoter from CdebDNAV and fcp terminator of a centric diatom Thalassiosira pseudonana; the latter contained zeocin resistance gene (ble) with the CdP1 promoter and fcp terminator from the pennate diatom Cylindrotheca fusiformis. Then, they were respectively transfected into a centric diatom Chaetoceros sp. and a pennate diatom Phaeodactylum tricornutum using microparticle bombardment methods. Transformant diatom cells were recovered in the presence of the respective antibiotics (nourseothricin or zeocin). Transformation efficiency was 1 transformant per 108 cells for Chaetoceros sp. and 5 per 108 cells for P. tricornutum. Successful integration of nat and/or ble genes into genomic DNA of the transformants was verified by means of Southern hybridization technique. These results indicate CdP1 promoter functions in both centric and pennate diatom species. As far as authors know, this is the first report describing the transfor mation system of marine diatom using viral promoter of marine algal virus. [Tomaru, Y., Shirai, Y., Suzuki, H., Nagumo, T., Nagasaki, K. (2008) Isolation and characterization of a novel single-stranded DNA virus infecting a cosmopolitan marine diatom Chaetoceros debilis. Aquat. Microb. Ecol. 50: 103-112.]