Fig. 3

The alas1 was the causative gene of the smu350 mutant. (A) Sequencing analysis revealed a 2-bp deletion within exon 7 of alas1 in CRISPR/Cas9 generated alas1 ^{Δ2/ Δ2} mutants. Uppercase sequences highlighted blue indicate exons; sequences in lowercase indicate introns. Sequences underlined in red indicate the CRISPR/Cas9 target in alas1. The red asterisk indicates the smu350 mutation site. Black boxes indicate the wild-type peptides, and the red box indicates the incorrect peptide generated by the altered splicing. The pink box indicates the pre-sequence domain (pfam09029). The green box indicates the aspartate aminotransferase superfamily (fold type I) domain of pyridoxal phosphate-dependent enzymes (cl18945). The box with zigzag edges indicates the truncated region. Black and red numbers denote distances to the start codon in wild type and mutants, respectively. (B) Alas1 protein was absent in alas1 ^{Δ2/ Δ2} mutants. Examination of Alas1 protein expression in the whole fish body by western blotting at 5 days post fertilization (dpf). GAPDH was used as the loading control. (C) The Sudan black B (SB) signal was absent in alas1 ^{Δ2/ Δ2} mutants. SB staining in siblings (upper) and alas1 ^{Δ2/ Δ2} mutants (lower) at 3 dpf. Boxed regions are magnified in the lower right-hand corner. Scale bars: 200 μm. (D) SB signal was absent in alas1^{smu350/ Δ2} mutants. SB staining in siblings (upper) and alas1^{smu350/ Δ2} mutants (lower) at 3 dpf. Boxed regions are magnified in the lower right-hand corner. Scale bars: 200 μm.