pdk-1 loss-of-function mutation sa709 is epistatic to zfp-1(ok554) and rde-4(ne299).

(A) Schematic of the pdk-1 gene with numbered boxes for exons and lines for introns, location of the sa709 mutation and predicted effects of the mutation on mRNA and protein; exons coding for the kinase domain and the pleckstrin homology domain (PH) according to [4] are indicated. (B) Expression levels of correctly spliced pdk-1 mRNA and intron 3-containing mRNA were determined by RT-qPCR, and ratios of spliced/unspliced isoforms in the indicated mutants were calculated and presented on a graph. The forward primer used spanned the exon 1/exon 2 junction, the reverse primer for the spliced isoform spanned the exon 3/exon 4 junction and the reverse primer for the intron 3-containing isoform was intron 3-specific (see Materials and Methods). (C) Intron 3-containing pdk-1 mRNA levels were measured by real time RT-qPCR in indicated mutants (L4 stage animals) and normalized to pdk-1(sa709). Results of two biological replicas are shown; error bars represent Standard deviation. (D, E) DAF-16::GFP nuclear localization in indicated mutants was assessed as described in Materials and Methods. Representative epifluorescence images of intestinal cells (D) were taken on a Zeiss AxioImager Z1 microscope at 630x total magnification; white arrowheads point to the nuclei.(A) 本图为pdk-1基因的示意图，以带有序号的方框表示外显子，线条代表内含子，标注了sa709突变的位点，以及该突变对mRNA和蛋白质的预期效应；依据文献[4]，标注了编码激酶结构域（kinase domain）与普列克底物蛋白同源结构域（pleckstrin homology domain, PH）的外显子区域。 (B) 通过逆转录实时定量PCR（RT-qPCR）检测了正确剪接的pdk-1 mRNA以及含内含子3的mRNA的表达水平；计算了指定突变体中剪接型/未剪接型转录本的比值并以图表展示。所用上游引物跨外显子1与外显子2的连接区域，针对剪接型转录本的下游引物跨外显子3与外显子4的连接区域，而针对含内含子3转录本的下游引物为内含子3特异性引物（详见材料与方法）。 (C) 通过实时逆转录定量PCR检测了指定突变体（L4期幼虫）中含内含子3的pdk-1 mRNA水平，并以pdk-1(sa709)作为参照进行标准化处理。展示了两次生物学重复的实验结果；误差棒代表标准差（Standard deviation）。 (D, E) 按照材料与方法中的描述，对指定突变体中DAF-16::绿色荧光蛋白（DAF-16::GFP）的核定位情况进行评估。肠道细胞的代表性落射荧光图像（D）通过蔡司AxioImager Z1显微镜以总放大630倍拍摄；白色箭头指向细胞核位置。