Construction of a germline-specific RNAi tool in C. elegans.
Construction of a germline-specific RNAi tool in C. elegans.
复制标题
线虫种系特异性 RNAi 工具的构建
DOI：
10.1038/s41598-019-38950-8
复制
发表时间：
2019
影响因子：
4.6
通讯作者：
Chen Di
中科院分区：
文献类型：
--
作者：
Zou Lina;Wu Di;Zang Xiao;Wang Zi;Wu Zixing;Chen Di
在多细胞生物中，分析复杂的生物功能通常需要组织特异性的遗传操作。秀丽隐杆线虫的生殖系不仅在生殖方面起调节作用，而且在代谢、应激反应和衰老方面也起调节作用。先前的研究使用rrf-1突变体作为种系特异性RNAi工具，rrf-1编码RNA定向RNA聚合酶。然而，rrf-1突变体在体细胞组织中表现出RNAi活性。在这里，我们将编码Argonaute蛋白的rde-1的indel突变与由sun-1种系特异性启动子驱动的单拷贝rde-1转基因结合在一起，构建了一种种系特异性RNAi菌株。生殖系RNAi的效率和特异性通过已知突变的RNAi表型、GFP报告基因表达的敲低以及RNAi敲低后组织特异性mrna的定量RT-PCR测量得到证实。与野生型动物相比，种系特异性RNAi菌株在繁殖、脂质积累、耐热性和寿命方面均无明显缺陷。通过筛选磷酸酶基因的RNAi亚库，我们发现了新的耐热性调节因子。我们共同创造了一个有用的工具，可以促进秀丽隐杆线虫种系特异性功能的遗传分析。
Analysis of complex biological functions usually requires tissue-specific genetic manipulations in multicellular organisms. The C. elegans germline plays regulatory roles not only in reproduction, but also in metabolism, stress response and ageing. Previous studies have used mutants of rrf-1, which encodes an RNA-directed RNA polymerase, as a germline-specific RNAi tool. However, the rrf-1 mutants showed RNAi activities in somatic tissues. Here we constructed a germline-specific RNAi strain by combining an indel mutation of rde-1, which encodes an Argonaute protein that functions cell autonomously to ensure RNAi efficiency, and a single copy rde-1 transgene driven by the sun-1 germline-specific promoter. The germline RNAi efficiency and specificity are confirmed by RNAi phenocopy of known mutations, knockdown of GFP reporter expression, as well as quantitative RT-PCR measurement of tissue-specific mRNAs upon RNAi knockdown. The germline-specific RNAi strain shows no obvious deficiencies in reproduction, lipid accumulation, thermo-tolerance and life span compared to wild-type animals. By screening an RNAi sub-library of phosphatase genes, we identified novel regulators of thermo-tolerance. Together, we have created a useful tool that can facilitate the genetic analysis of germline-specific functions in C. elegans.