Cope’s gray treefrog, Dryophytes chrysoscelis, withstands the physiological challenges of corporeal freezing, partly by accumulating cryoprotective compounds of hepatic origin, including glycerol, urea, and glucose. We hypothesized that expression of genes related to cryoprotectant mobilization and stress tolerance would be differentially regulated in response to cold. Using high-throughput RNA sequencing (RNA-Seq), a hepatic transcriptome was generated for D. chrysoscelis, and gene expression was compared among frogs that were warm-acclimated, cold-acclimated, and frozen. A total of 159,556 transcripts were generated; 39% showed homology with known transcripts, and 34% of all transcripts were annotated. Gene-level analyses identified 34,936 genes, 85% of which were annotated. Cold acclimation induced differential expression both of genes and non-coding transcripts; freezing induced few additional changes. Transcript-level analysis followed by gene-level aggregation revealed 3582 differentially expressed genes, whereas analysis at the gene level revealed 1324 differentially regulated genes. Approximately 3.6% of differentially expressed sequences were non-coding and of no identifiable homology. Expression of several genes associated with cryoprotectant accumulation was altered during cold acclimation. Of note, glycerol kinase expression decreased with cold exposure, possibly promoting accumulation of glycerol, whereas glucose export was transcriptionally promoted by upregulation of glucose-6-phosphatase and downregulation of genes of various glycolytic enzymes. Several genes related to heat shock protein response, DNA repair, and the ubiquitin proteasome pathway were upregulated in cold and frozen frogs, whereas genes involved in responses to oxidative stress and anoxia, both potential sources of cellular damage during freezing, were downregulated or unchanged. Our study is the first to report transcriptomic responses to low temperature exposure in a freeze-tolerant vertebrate. The hepatic transcriptome of Dryophytes chrysoscelis is responsive to cold and freezing. Transcriptomic regulation of genes related to particular pathways, such as glycerol biosynthesis, were not all regulated in parallel. The physiological demands associated with cold and freezing, as well as the transcriptomic responses observed in this study, are shared with several organisms that face similar ecophysiological challenges, suggesting common regulatory mechanisms. The role of transcriptional regulation relative to other cellular processes, and of non-coding transcripts as elements of those responses, deserve further study.

中文翻译：

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耐冻的Cope的灰色树蛙Dryophytes chrysoscelis的肝转录组：对冷驯化和冷冻的反应

Cope的灰色树蛙Dryophytes chrysoscelis可以承受有形冷冻的生理挑战，部分是通过积累肝脏起源的冷冻保护性化合物（包括甘油，尿素和葡萄糖）来进行的。我们假设与低温保护剂动员和压力耐受性相关的基因表达将在寒冷时得到差异调节。使用高通量RNA测序（RNA-Seq），产生了金黄色葡萄球菌的肝转录组，并比较了在温驯，冷驯化和冷冻的青蛙中的基因表达。总共产生了159,556个成绩单；39％的人与已知的成绩单具有同源性，所有成绩单的34％都带有注释。基因水平分析鉴定了34,936个基因，其中85％带有注释。冷驯化诱导基因和非编码转录本的差异表达；冻结导致很少的其他变化。转录物水平分析，然后是基因水平聚集，揭示了3582个差异表达的基因，而在基因水平的分析则揭示了1324个差异调节的基因。大约3.6％的差异表达序列是非编码的，没有可识别的同源性。与冷保护剂积累相关的几个基因的表达在冷驯化过程中发生了变化。值得注意的是，甘油激酶表达随着冷暴露而降低，可能促进甘油的积累，而葡萄糖输出通过葡萄糖-6-磷酸酶的上调和各种糖酵解酶基因的下调而在转录上得到促进。与热休克蛋白反应有关的几个基因，在寒冷和冰冻的青蛙中，DNA修复和泛素蛋白酶体途径被上调，而在冰冻过程中潜在的细胞损伤源-氧化应激和缺氧反应中涉及的基因被下调或保持不变。我们的研究是第一个报告耐冻脊椎动物中低温暴露的转录组反应的研究。干燥水晶藻的肝转录组对冷和冰冻有反应。与特定途径相关的基因（如甘油生物合成）的转录组调控并非全部并行进行。与寒冷和冰冻相关的生理需求，以及在这项研究中观察到的转录组反应，都与面临相似生态生理挑战的几种生物体共享，提示了共同的调节机制。