热适应差异表达基因消减文库的筛选

PCR-select differential screening of a subtracted cDNA library of heat-adapted rat liver

目的:研究热适应大鼠的肝细胞基因差异表达,探讨热适应的分子机制。方法:构建热适应差异表 达基因消减文库,扩增并克隆T／A载体形成重组质粒,转化感受态细胞后分离获得目的基因,采用PCR-select dif ferential screening技术筛选差异表达基因,测序后所得序列与GenBank中的已知序列进行比对,确定其可能的功能。 结果:确认8个上调表达基因,与GenBank中已知序列进行比较,证实其中有3种已知基因,5个新序列表达标签 (EST)。结论:相关基因表达水平上调可能参与生物体热适应的信号转导通路,而p53可能是热适应分子环路的又一 节点。

AIM： To reveal the molecular mechanisms of heat adaptation by study the change of gene expression in rat liver when the rats exposed to hyperthennia stress and heat adaptation. METHODS： Differentially expressed genes in rat liver in association with heat adaptation were identified using the suppression subtractive hybridization （SSH） technique to prepare subtracted eDNA libraries in heat adaptation. The PCR- amplified eDNA fragments generated by SSH were then ligated into the plasmid pGEM - T （Promega）. PCR - select differential screening technique was used to filtrate differentially expressed genes. Each of these dif- ferentially expressed genes was sequenced and was compared to known sequences in the GenBank database. RESULTS： eight upregulating expressed genes segments were isolated from the up - regulating differentially expressed library, three of which were known gene such as p53 and five genes were new expressed sequence tags. CONCLUSION： Up- regulating expressed genes such as 1353 maybe participate in the signal transduction pathway of heat adaptation.