超嗜热古菌Sulfolobus tokodaii RadA蛋白的克隆表达及 其辐射可诱导性

Cloning, expression and radiation inducibility of RadA from the hyperther-mophilic archaeon Sulfolobus tokodaii

摘要:

RecA/Rad51/RadA家族蛋白是细胞内重要的重组修复蛋白, 在功能上非常保守。研究发现在细菌、真核生物、甲烷古菌和嗜盐古菌细胞内RecA/Rad51/RadA均可以受紫外线辐射诱导转录。而对极端嗜热古菌中的RadA辐射可诱导性仍存在争议。通过体外表达极端嗜热古菌Sulfolobus tokodaii的RadA蛋白, 制备抗体, 利用免疫学方法并结合RT-PCR分析, 对嗜热古菌S. tokodaii中RadA的辐射诱导进行了研究。经过100J/m2和200J/m2 UV辐照处理, radA基因的转录分别上调了2倍和3倍, 同时RadA蛋白的表达分别上升了1.5倍和1倍。实验结果表明S. tokodaii中RadA可以被紫外线辐射诱导表达, 证实了极端嗜热古菌S. tokodaii细胞中存在DNA损伤诱导反应的观点。

关键词:嗜热古菌; RadA; 辐射可诱导性; DNA修复

Abstract:

RecA/Rad51/RadA recombinases are important recombination proteins with conserved functions. Studies on the enzymes have shown that members of RecA/Rad51/RadA family from bacteria, eukaryota, methanogens and halophilic archaea have UV inducibility. However, the UV inducibility of RadA homologues from hyperthermophilic archaea is controversial.We analyzed the UV inducibility of Sulfolobus tokodaii RadA by RT-PCR and immune assays. Comparing with the mock, the transcription and expression of the radA increased 2 and 1.5 folds respectively after UV irradiation at 100 J/m2, or 3 and 1 fold at 200 J/m2. These results demonstrated that S. tokodaii RadA could be induced after UV treatment. In addition, pro-teome induction analysis proved that there existed a DNA damage induction response in S. tokodaii, which further supported RadA inductility in this hyperthermophilic archaeon.

Key words:hyperthermophilic archaea; RadA; radiation inducibility; DNA repair