PDF(Pubmed)
Abstract：
Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.
摘要：
单细胞真核生物代表着巨大的进化多样性。然而,这种多样性背后的分子机制在很大程度上仍未被探索，部分原因是遗传工具仅限于少数模型物种。草履虫是一种众所周知的单细胞真核生物，具有出乎意料的大种系基因组，其中只有2%保留在性过程后的体细胞基因组中，揭示了广泛的DNA消除。然而,由于缺乏合适的遗传工具，阻碍了对控制这一过程的分子机制的进一步理解。这里,我们报道了成功应用基因敲除和蛋白质定位方法来研究铜绿假单胞菌中管家基因和发育调节基因的功能。使用这些方法，我们通过喂食获得了预期的RNAi表型，并通过显微注射含有荧光蛋白或抗体标签的融合构建体确定这些蛋白的定位。最后,我们用这些方法揭示了加拿大猪猪，驯化的piggyBac转座酶,对于性发育至关重要，并且可能是直接参与DNA切割的活性转座酶。这些方法的应用为今后研究紫草基因功能奠定了基础，可用于回答未来重要的生物学问题。
