Figure 3
From: Overexpression of Tisochrysis lutea Akd1 identifies a key cold-induced alkenone desaturase enzyme
Characterization of the TOD-1 overexpressing mutants of Tisochrysis lutea. (a) The expression constructs containing TOD-1 and PyAph7 (a codon-modified hygromycin B-resistance gene optimised for GC-rich algae29). Double-headed arrows: regions amplified in the genotyping PCR reactions. Lhcf17pro: endogenous promoter for Light-harvesting complex f17 (Lhcf17) gene of T. lutea. CrRbcSter: terminator from the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit gene from Chlamydomonas reinhardtii. (b) Genotyping by genomic PCR. WT: wild type. R11-10: a PyAph7-harbouring control strain. AtOM 1 [R25-6] and 2 [R26-2]: alkenone desaturase candidate overexpression mutants transformed with vectors pLf17A7-TOD-1-R and -F, respectively. (c) Growth profiles of the different strains. (d) Expression analysis of TOD-1. Relative expression was normalised to the value in WT strain on day 1. Data: mean ± s.d. (n = 3) except for AtOM 1 on days 9 (n = 2) and 13 (n = 1) and AtOM 2 on day 3 (n = 2). Significant differences determined by statistical analysis (ANOVA-Tukey-Kramer) are shown (*p < 0.01 and **p < 0.025). (e) Partial GC-FID chromatograms on day 13. (f) Total C37-alkenone contents. Peak numbers and colours are same as shown in Fig. 1a. (g) C37:3/C37:2 ratios. Data: mean ± s.d. (n = 3). (h) Relative C37:3/C37:2 ratios normalised to that in WT. Original unmodified images of gel electrophoresis are shown in Supplementary Fig. 7.
