somatostatin1基因突变斑马鱼仔鱼转录组分析

Transcriptome sequencing analysis of somatostatin 1 gene mutation in zebrafish larvae

摘要:

利用CRISPR/Cas9基因编辑技术在斑马鱼中构建了可稳定遗传的生长抑素（somatostatin，SST）基因（sst1）突变体鱼系。通过比较6 dpf（days post fertilization，dpf）的sst1^-/-突变体和野生型仔鱼转录组，发现sst1^-/-突变体相关生化过程和信号通路发生了显著变化。相比野生型，sst1^-/-突变体仔鱼中有354个基因显著上调，504个基因显著下调。GO（gene ontology）富集分析表明，大部分差异基因与氨基酸和蛋白质生物合成相关。KEGG（kyoto encyclopedia of genes and genomes）通路富集显示代谢通路、氨基酸合成、氨基酰基-tRNA合成、核糖体合成和rRNA加工相关通路表达增强。结果表明，sst1^-/-突变体蛋白质生物合成和代谢过程更加活跃。RT-qPCR验证结果显示，随机选取的10个基因mRNA表达变化趋势与转录组测序结果一致，说明转录组测序结果真实可靠。6月龄突变体体质量、体长和野生型相比并无显著差异。本研究结果为进一步挖掘生长抑素基因家族的潜在功能奠定基础。

Abstract:

Somatostatin (SST), is a kind of neurohormone with key roles in regulating growth and metabolism in all vertebrates. In zebrafish and other fishes, SST peptides are encoded by six genes, and sst1 exists in all vertebrates and is the most evolutionarily conservative. Here, we explored sst functions through a transcriptomic comparison of 6 days post fertilization (dpf) zebrafish sst1^-/- mutants generated by CRISPR/Cas9 gene-editing technology and their wild type counterparts. It is found that several biological processes and biochemical pathways were modified in sst1^-/- zebrafish. The mutant had 354 significantly up-regulated and 504 significantly down-regulated genes. Gene ontologyi(GO) enrichment analysis showed that most of the differentially expressed genes were related to the biosynthesis of amino acids and proteins. Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment indicated metabolic pathways as well as pathways related to amino acid biosynthesis, aminoacyl-tRNA biosynthesis, ribosome biogenesis and rRNA processing were highly represented in the mutant, indicating a more active protein biosynthesis and metabolism. The results of RT-qPCR illustrated that the mRNA expression trends of 10 randomly selected genes were consistent with the results of transcriptome sequencing analysis, indicating that the transcriptome results were accurate and reliable. There was no significant difference in body weight and body length between 6-month-old mutant and wild-type. The results provide a foundation for further exploring the potential functions of the somatostatin gene family.