Isolation of several anti-stress genes from the halotolerant green alga Chlamydomonas by simple functional expression screening with Escherichia coli

To isolate anti-stress genes from a halotolerant marine green alga, a simple screening method based on the bacterial expression system was examined. The method consisted of three steps: (i) directional cDNA library construction in a lambda ZAPII vector, (ii) in vivo mass excision of a lambda ZAPII library into phagemid DNA, and (iii) screening for anti-salt-stress and anti-oxidative-stress genes by culturing bacterial cells carrying the in vivo excised phagemid on selection agar plates with a high concentration of NaCl and/or methyl viologen (MV), and by isolating stress-tolerant bacterial colonies. By this method, we screened the cDNA library of halotolerant Chlamydomonas sp. strain W-80, and isolated several stress-related gene homologs, such as glutathione peroxidase, ascorbate peroxidase, bbc1 (breast basic conserved; a low temperature induced gene in Brassica napus), and cyanide-resistant alternative oxidase.