SupportingInformation.MaterialsandMethods分析.docVIP

Supporting Information. Materials and Methods Strains and culture conditions Chlamydomonas reinhardtii wild-type strains CC400 (mt-), 137c (mt+) and the mutant pgr5 (derived from 137c; Dent et al., 2005) were obtained from the Chlamydomonas Center (www.C). The pgrl1 mutant was obtained from Gilles Peltier. The hpm91 mutant was isolated through a mutant screen as described below. The algal cells were cultured in TAP or in high-salt minimal (HSM) medium at 25 °C under 60 μmol m-2 s-1 continuous light. Growth was measured as OD750 at different times of cultivation. For protein analysis, cells were harvested by centrifugation at 2500 g and 4 °C for 5 min. Cell pellets were washed once with 0.02 M Hepes/KOH buffer (pH 7.5) and stored at -70 °C. Mutant generation and isolation RNAi /miRNA cell lines targeted for FNR or PGRL1 silencing were produced as previously (Zhao et al. 2013) using the pChlamiRNA3 vector obtained from the Chlamydomonas Center. Target gene-specific oligonucleotides were designed using the WMD3 software (./cgi-bin/webapp.cgi) and verified using the expressed sequence tag database?(http://est.kazusa.or.jp/en/plant/chlamy/EST/blast. html). Plasmids were constructed by annealing oligonucleotides that target the 3’‐untranslated region of the?PGRL1?or FNR genes followed by ligation into the vector at the unique?SpeI site located in the amiRNA precursor. The cell wall-deficient strain CC400 (wild type) was co-transformed with the two KpnI- linearized plasmids pChlamiRNA int3 containing the FNR and PGRL1 targeting sequences with the APHVIII gene which confers paromomycin resistance using the glass bead method (Kindle 1990). After selection on TAP plates with 10 μg/ml paromomycin (Sigma), insertion mutants were screened for altered H2 evolution by gas chromatography as described (Sun et al. 2013). Among the mutants with the highest yield of H2 production, hpm91 was chosen for subsequent characterization. Determination of H2 and O2 production Induction and a