Establishing RNAi in a Non-Model Organism: The Antarctic Nematode <i>Panagrolaimus</i> sp. DAW1

The Antarctic nematode Panagrolaimus sp. DAW1 is one of the only organisms known to survive extensive intracellular freezing throughout its tissues. Although the physiological mechanisms of this extreme adaptation are partly understood, the molecular mechanisms remain largely unknown. RNAi is a method that allows the examination of gene function in a direct, targeted manner, by knocking out specific mRNAs and revealing the effects on the phenotype. In this study we have explored the viability of RNAi in Panagrolaimus sp. DAW1. In the first trial, nematodes were fed E. coli expressing Panagrolaimus sp. DAW1 dsRNA of the embryonic lethal genes rps-2 and dhc, and the blister gene duox. Pd-rps-2(RNAi)-treated nematodes showed a significant decrease in larval hatching. However, qPCR showed no significant decrease in the amount of rps-2 mRNA in Pd-rps-2(RNAi)-treated animals. Several soaking protocols for dsRNA uptake were investigated using the fluorescent dye FITC. Desiccation-enhanced soaking showed the strongest uptake of FITC and resulted in a significant and consistent decrease of mRNA levels of two of the four tested genes (rps-2 and tps-2a), suggesting effective uptake of dsRNA-containing solution by the nematode. These findings suggest that RNAi by desiccation-enhanced soaking is viable in Panagrolaimus sp. DAW1 and provide the first functional genomic approach to investigate freezing tolerance in this non-model organism. RNAi, in conjunction with qPCR, can be used to screen for candidate genes involved in intracellular freezing tolerance in Panagrolaimus sp. DAW1.南极线虫Panagrolaimus sp. DAW1是目前已知少数可在全身组织发生广泛细胞内结冰的生物之一。尽管这种极端适应的生理机制已得到部分阐明，但其分子调控机制仍大多未被揭示。RNA干扰（RNA interference, RNAi）是一种可通过靶向敲除特定信使RNA（messenger RNA, mRNA），以直接、精准的方式研究基因功能并观察其对表型影响的实验技术。本研究探究了RNAi技术在Panagrolaimus sp. DAW1中的适用性。在初始试验中，研究人员用表达Panagrolaimus sp. DAW1胚胎致死基因rps-2、dhc以及水疱基因duox的双链RNA（double-stranded RNA, dsRNA）的大肠杆菌（Escherichia coli, E. coli）喂食该线虫。经Pd-rps-2(RNAi)处理的线虫，其幼虫孵化率出现显著下降。然而实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qPCR）检测结果显示，经Pd-rps-2(RNAi)处理的线虫体内rps-2的mRNA水平并无显著降低。研究人员以荧光染料异硫氰酸荧光素（fluorescein isothiocyanate, FITC）为标记，探究了多种可促进该线虫摄取dsRNA的浸泡方案。其中，干燥增强浸泡法的FITC摄取效果最强，且能使4个待测基因中的2个（rps-2与tps-2a）的mRNA水平出现显著且稳定的下调，表明该线虫可有效摄取携带dsRNA的溶液。上述结果证实，基于干燥增强浸泡法的RNAi技术在Panagrolaimus sp. DAW1中具备可行性，同时也为这种非模式生物的细胞内结冰耐受机制研究提供了首个功能基因组学研究手段。结合qPCR技术，RNAi可用于筛选Panagrolaimus sp. DAW1中与细胞内结冰耐受相关的候选基因。