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Plant and Cell Physiology Advance Access originally published online on February 28, 2008
Plant and Cell Physiology 2008 49(4):625-632; doi:10.1093/pcp/pcn039
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Expression of Exogenous Genes Under the Control of Endogenous HSP70 and CAB Promoters in the Closterium peracerosum–strigosum–littorale complex

Jun Abe1,*, Yuji Hiwatashi2,3, Motomi Ito4, Mitsuyasu Hasebe2,3 and Hiroyuki Sekimoto1,*

1Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo, 112-8681 Japan
2Division of Evolutionary Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan
3School of Life Science, The Graduate University for Advanced Studies, Okazaki, 444-8585 Japan
4Department of General Systems Studies, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, 153-8902 Japan

*Corresponding authors: Hiroyuki Sekimoto, E-mail, sekimoto{at}fc.jwu.ac.jp; Fax, + 81-3-5981-3674; Jun Abe, E-mail, junabe{at}fc.jwu.ac.jp; Fax, + 81-3-5981-3674.


  Abstract

A unicellular charophyte alga, Closterium peracerosum–strigosum–littorale complex (C. psl. complex), has been studied in order to obtain basic information regarding sexual reproduction in plants. Systems for gene introduction and transient expression were developed for endogenous genes using phleomycin resistance (ble) and Chlamydomonas green fluorescent protein (cgfp) genes as selection markers. These genes have codon usage similar to that of genes in the C. psl. complex. To drive these genes strongly into C. psl. complex cells, two native promoters of the C. psl. complex genome—CpHSP70 and CpCAB1—were linked to a ble::cgfp fusion gene and introduced into the cells by particle bombardment. Following 2 d of incubation, we found 500 cells expressing GFP under the control of the CpHSP70 promoter, which were identified following heat shock treatment at 42°C, and 100 cells expressing GFP under the control of the CpCAB1 promoter, which were observed in lit conditions. In contrast, the GFP signal was only detected in two cells when ble::cgfp under control of the cauliflower mosaic virus 35S promoter was introduced. The ble::cgfp fusion protein was detected in the nucleus, whereas the single cgfp protein was detected in the cytoplasm. Our results indicate that the newly isolated native promoters of CpHSP70 and CpCAB1 are useful tools for inducing exogenous gene expression under heat shock and lit conditions, respectively. In addition, this strategy can be used for transient assays, such as the intracellular localization of unknown gene products in the C. psl. complex.

Keywords: Charophyte alga — Closterium — Exogenous gene expression — GFP — Native promoter — Particle bombardment

Abbreviations: ble, phleomycin resistance gene; CAB, chlorophyll a/b-binding protein gene; CaMV, cauliflower mosaic virus; cgfp, GFP gene adapted to codon usage of C. reinhardtii; C. psl. complex, Closterium peracerosum–strigosum–littorale complex; EST, expressed sequence tag; GFP, green fluorescent protein; GUS, β-glucuronidase; HSP70, heat shock protein 70 gene; TAIL-PCR, thermal asymmetric interlaced PCR.

(Received October 13, 2007; Accepted February 21, 2008)
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