Abstract
A microalga Botryococcus braunii, race B produces a large amount of liquid hydrocarbons which can be used as fuel of airplanes. However, its gene manipulation is to be developed for its commercial applications because of its very slow growth. In order to establish transformation technology in this alga, we first surveyed appropriate drugs for selecting transformants. Algal cells were cultured in liquid Chu13 medium under a continuous illumination at 2500 lux and with 2.5% CO2. Aliquots of the liquid culture containing 4.5 x104 cells were inoculated on Chu13 agar plates supplemented with various concentrations of an antibiotic. Green algal colonies were observed on an agar plate containing no drugs after three-week culture, while addition of 10, 20 mg/l of hygromycin, 50, 100 mg/l of spectinomycin, or 6 mg/l of zeocin was inhibitory to the growth. Effects of treatment with cell wall degrading enzymes on antibiotic sensitivities are also studied. For the isolation of promoters to express exogenous genes in the alga, we are also amplifying 5'-upstream regions of genomic fragments of HSP70, small subunit of Rubisco and squalene synthase by RESDA-PCR.
