强紫外线辐射生境中南极冰藻光修复酶及其修复DNA紫外辐射损伤分子机制研究

臭氧破坏导致紫外线辐射增强，对生物和人影响日益显著。目前大部分研究关注其对生物群落和个体的影响与危害，而很少探究南极强紫外线辐射生境中生物的极端生存机制，尤其未从DNA紫外损伤与修复关键科学问题上认识其强紫外线辐射环境适应分子机制。本研究选取南极关键物种冰藻，基于已完成冰藻Chlamydomonas sp.ICE-L紫外胁迫转录组测序与分析，且RACE出其CPD光修复酶全长基因，拟采用免疫印迹等技术探明冰藻DNA紫外线辐射损伤形成嘧啶二聚体分子机制；应用RT-PCR等技术了解冰藻光修复酶基因表达与调控机理；构建冰藻光修复酶基因重组大肠杆菌和酵母，验证其体内活性；选用HPLC等方法纯化表达产物，表征理化特性，检测体外活性；运用基因定点突变和基因敲除等方法，了解其必需结构基团，验证其生理功能。据此，将从基因及蛋白水平认识冰藻适应南极强紫外线辐射分子机理，探讨其对人类紫外线辐射损伤防治重要意义。

The depletion of ozone increases the biologically harmful solar ultraviolet (UV) radiation, which leads to a variety of adverse effects to organisms and human being. Although extensive research has been conducted on the UV damage to the biotic community and the individual, few studies have focused on the adaptations of polar organisms to UV radiation so far; especially in terms of molecular mechanism of DNA damage and repair. Based on transcriptome sequencing and analyses of Antarctic keystone specie Chlamydomonas sp. ICE-L under UV stress, RACE and qRT-PCR were performed to obtain the full-length DNA photolyase gene and determine the effects of UV radiation on the expression levels,respectively. With the cDNA introducing in Escherichia coli and Saccharomyces cerevisiae, in vivo photolyase activity assays was performed. Then the photolyase was overproduced and purified by HPLC. To assess the contribution of the photolyase to the photorepair of UV-induced DNA damage, western blot and in vitro photolyase activity assays was performed. Gene mutation and knocked out will be performed to understand its essential gene and physiological function. Therefore, in this study we focused on the molecular mechanism of Chlamydomonas sp. ICE-L under UV resistance in both gene and protein levels, and this will accelerate the utilization of photolyase in human being UV damage.