Transcriptomics of rapid cold-hardening and cold shock recovery in Sarcophaga bullata

The ability to rapidly respond to changes in temperature is critical for insects and other ectotherms living in variable environments. In a physiological process termed rapid cold-hardening (RCH), exposure to non-lethal low temperature allows many insects to significantly increase their cold tolerance in a matter of minutes to hours. Additionally, there are rapid changes in gene expression and cell physiology during recovery from cold injury, and we hypothesize that RCH may modulate some of these processes during recovery. In this study, we used a cDNA microarray to examine the molecular mechanisms of RCH and cold shock (CS) recovery in the flesh fly, Sarcophaga bullata. With our custom 2-color array, we measured expression of ~15,000 ESTs during RCH and during recovery from cold shock. Surprisingly, no transcripts were upregulated during RCH, and likewise, RCH had a minimal effect on the transcript signature during recovery from cold shock. However, during recovery from cold shock, we observed differential expression of ~1,400 ESTs, including a number of heat shock proteins, cytoskeletal components, and genes from several cell signaling pathways. Several gene pathways correlated well with metabolomics data, indicating that coordinated changes in gene expression and metabolism contribute to recovery from cold shock. Four treatment groups (C, RCH, CS+2R, RCH+CS+2R), four biological replicates of four pooled individuals for each treatment. Each phenotype was hybridized with the control, and the CS+2R and RCH+CS+2R groups were also hybridized together.

对于生活在多变环境中的昆虫及其他外温动物而言，快速响应温度变化的能力至关重要。在被称为快速冷驯化（rapid cold-hardening, RCH）的生理过程中，暴露于非致死低温环境可使多数昆虫在数分钟至数小时内显著提升其耐寒能力。此外，昆虫从冷损伤中恢复的过程中会伴随基因表达与细胞生理的快速变化，我们推测快速冷驯化或许会在恢复阶段调控其中部分过程。本研究以肉蝇（Sarcophaga bullata）为实验材料，采用cDNA微阵列技术探究快速冷驯化与冷激（cold shock, CS）恢复的分子机制。依托定制化双色微阵列，我们检测了快速冷驯化阶段以及冷激恢复阶段中约15000条表达序列标签（expressed sequence tags, ESTs）的表达水平。令人意外的是，快速冷驯化阶段未检测到任何转录本上调；同样，快速冷驯化对冷激恢复阶段的转录特征影响极小。但在冷激恢复阶段，我们观测到约1400条表达序列标签存在差异表达，其中涵盖多种热休克蛋白、细胞骨架组分以及多条细胞信号通路相关基因。部分基因通路与代谢组学数据呈现显著相关性，这表明基因表达与代谢的协同变化助力冷激后的机体恢复。本研究共设置4个处理组（C、RCH、CS+2R、RCH+CS+2R），每个处理设置4个生物学重复，每个重复包含4只混合的个体。所有表型样本均与对照样本进行杂交，同时CS+2R组与RCH+CS+2R组之间也开展了杂交实验。