Gene expression analysis of third instar larvae ski3 mutants

Purpose: To identify genes that are differentially expressed between control and ski3 mutants Methods: Gene expressions of control and ski3 mutants of third instar larvae were generated by deep sequencing, in two replicates, using Illumina NovaSeq 6000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Bowtie2 and TopHat followed by Cufflinks. Results: We mapped 800,000-1,200,000 sequence reads per each sample to the fruit fly genome (BDGP6) and identified 93,606 transcripts in the whole body of control and ski3[f03251] with TopHat workflow. Analysis of RNA-seq data idnetified 32 differentially expressed genes relevant to mitochondrial function between control and ski3 mutants with FDR <0.05. The mutation affected over 600 genes, which are involved in many different cellular functions. The results were complex: among 32 affected genes, half of them were upregulated, and the remaining half were downregulated. Six genes were related to the TCA cycle. Of these, the downregulated genes include CG9582 (alpha-ketoglutarate transmembrane transporter), CG32832 (mitochondrial pyruvate carrier), and CG7514 (oxoglutarate:malate antiporter). On the other hand, upregulated genes include AcCoAS (Acetyl-CoA synthase), Sirup (Succinate dehydrogenase), and Men-b (malate dehydrogenase), all of which were directly involved in the TCA cycle. Conclusions: Our study represents the first detailed analysis of ski3 mutant transcriptomes, with biologic replicates, generated by RNA-seq technology. In order to examine the effects of ski3 mutations on gene expression, we performed RNA-seq analyses of wild-type and mutant larvae. Downregulation of transporter, carrier and antiporter suggested a chronic undersupply of TCA cycle substrates, while some of the TCA cycle enzymes were upregulated. Overall design: Whole body mRNA profiles of third instar larvae control (w1118) and ski3[f03251] homozygotes were generated by deep sequencing, in two replicates, using Illumina NovaSeq 6000.

研究目的：鉴定对照组与ski3突变体之间存在显著表达差异的基因。
实验方法：采用Illumina NovaSeq 6000测序平台，对三龄幼虫的对照组及ski3突变体进行双生物学重复的RNA深度测序，以获得基因表达谱数据。对通过质量过滤的序列读段，采用两种方法在转录本异构体层面开展分析：先通过Bowtie2与TopHat进行序列比对，再结合Cufflinks完成后续转录本分析。
研究结果：将每个样本的800000~1200000条序列读段比对至果蝇基因组（BDGP6），并通过TopHat分析流程，在对照组与ski3[f03251]的全虫体中鉴定出93606个转录本。对RNA-seq数据的分析显示，在错误发现率（False Discovery Rate, FDR）<0.05的筛选标准下，共鉴定出32个与线粒体功能相关的差异表达基因。该突变影响了超过600个参与多种不同细胞功能的基因。研究结果呈现出显著复杂性：在这32个受影响的基因中，半数呈现上调表达，另一半则呈现下调表达。其中6个基因与三羧酸循环（TCA cycle）相关。下调的差异基因包括CG9582（α-酮戊二酸跨膜转运蛋白）、CG32832（线粒体丙酮酸载体蛋白）以及CG7514（氧戊二酸:苹果酸反向转运蛋白）；而上调的差异基因则包括AcCoAS（乙酰辅酶A合成酶）、Sirup（琥珀酸脱氢酶）以及Men-b（苹果酸脱氢酶），上述基因均直接参与三羧酸循环。
研究结论：本研究首次借助RNA-seq技术结合生物学重复样本，对ski3突变体的转录组开展了详细解析。为探究ski3突变对基因表达的影响，我们对野生型与突变体幼虫进行了RNA-seq分析。转运蛋白、载体蛋白及反向转运蛋白的下调表达，提示TCA循环底物存在慢性供应不足的情况；而部分TCA循环酶则呈现上调表达特征。
实验整体设计：采用Illumina NovaSeq 6000测序平台，对三龄幼虫的对照组（w1118）与ski3[f03251]纯合突变体进行双生物学重复的mRNA深度测序，以获取全虫体的mRNA表达谱。