Cold hardiness is fundamental for amphibians to survive during the extremely cold winter on the Qinghai-Tibet plateau. Exploring the gene regulation mechanism of freezing-tolerant Rana kukunoris could help us to understand how the frogs survive in winter. Transcriptome of liver and muscle of R. kukunoris collected in hibernation and spring were assisted by single molecule real-time (SMRT) sequencing technology. A total of 10,062 unigenes of R. kukunoris were obtained, and 9,924 coding sequences (CDS) were successfully annotated. Our examination of the mRNA response to whole body freezing and recover in the frogs revealed key genes concerning underlying antifreeze proteins and cryoprotectants (glucose and urea). Functional pathway analyses revealed differential regulated pathways of ribosome, energy supply, and protein metabolism which displayed a freeze-induced response and damage recover. Genes related to energy supply in the muscle of winter frogs were up-regulated compared with the muscle of spring frogs. The liver of hibernating frogs maintained modest levels of protein synthesis in the winter. In contrast, the liver underwent intensive high levels of protein synthesis and lipid catabolism to produce substantial quantity of fresh proteins and energy in spring. Differences between hibernation and spring were smaller than that between tissues, yet the physiological traits of hibernation were nevertheless passed down to active state in spring. Based on our comparative transcriptomic analyses, we revealed the likely adaptive mechanisms of R. kukunoris. Ultimately, our study expands genetic resources for the freezing-tolerant frogs.

中文翻译：

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比较转录组分析揭示了青藏高原林蛙对寒冷胁迫的适应策略

抗寒能力是两栖动物在青藏高原严寒冬季生存的基础。探索耐冻林蛙的基因调控机制可以帮助我们了解青蛙如何在冬季生存。利用单分子实时（SMRT）测序技术辅助采集冬眠和春季的库库诺丽丝肝脏和肌肉转录组。总共获得了10,062个R. kukunoris unigenes，并成功注释了9,924个编码序列（CDS）。我们对青蛙全身冷冻和恢复的 mRNA 反应的检查揭示了与潜在抗冻蛋白和冷冻保护剂（葡萄糖和尿素）有关的关键基因。功能途径分析揭示了核糖体、能量供应和蛋白质代谢的差异调节途径，显示出冷冻诱导的反应和损伤恢复。与春蛙肌肉相比，冬蛙肌肉中与能量供应相关的基因表达上调。冬眠青蛙的肝脏在冬季保持适度的蛋白质合成水平。相比之下，肝脏在春季进行密集的高水平蛋白质合成和脂质分解代谢，产生大量新鲜蛋白质和能量。冬眠和春季之间的差异小于组织之间的差异，但冬眠的生理特征在春季仍然传递到活跃状态。基于我们的比较转录组分析，我们揭示了 R. kukunoris 可能的适应机制。最终，我们的研究扩大了耐冻青蛙的遗传资源。