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

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.

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。

本项目通过基因克隆得到南极衣藻Chlamydomonas sp. ICE-L光修复酶基因CPD，并对该基因进行生物信息学分析；建立了CPD光修复酶原核和真核异源表达体系，并对原核和真核表达条件进行了优化；对原核和真核表达的蛋白进行了纯化和结构鉴定，并验证了南极衣藻光修复酶蛋白的体内和体外活性。项目通过基因工程手段构建南极冰藻CPD光修复酶基因表达载体，获得高效表达基因工程菌株，建立1套基因工程产品中试生产工艺技术路线，并建设南极冰藻CPD光修复酶中试生产线1条，完成了紫外线防护及紫外线损伤修复类化妆品应用试验，试用效果良好，项目产业化后将实现重要经济社会效益。主要成果总结如下：1、通过普通PCR和RACE法克隆得到南极衣藻Chlamydomonas sp.ICE-L的光修复酶基因CPD，其cDNA全长2651bp，编码579个氨基酸，预测蛋白相对分子质量为64.3KDa，等电点为8.982；经同源性对比，发现其CPD氨基酸序列与莱茵衣藻CPD的相似度为68%；2、通过实时荧光定量PCR技术对南极衣藻CPD基因在非生物胁迫条件（光强、紫外）下的差异表达进行相对定量分析。在紫外胁迫条件下，CPD基因表达量先有稍微降低，进而大幅升高，并在4h达到最大值，这说明CPD基因对紫外胁迫是敏感的，进而保护自身和维持正常的生理功能。3、成功构建CPD光修复酶基因大肠杆菌原核表达载体，并在菌株BL21（DE3）中诱导表达。同时实现了南极衣藻CPD光修复基因在酿酒酵母Saccharomyces cerevisiae SCPD中成功表达。4、利用Ni-NTA亲和层析对光修复酶目的蛋白进行分离纯化，得到纯CPD光修复酶蛋白；利用Edman降解法对光修复酶蛋白进行N端测序，显示该蛋白N端到C端依次为M-P-K-R-T，与目标序列 MPKRT相符，证实为CPD光修复酶；对表达CPD光修复酶的体内外活性研究表明，CPD光修复酶能够快速对紫外辐射损伤做出反应，高效修复嘧啶二聚体，说明CPD光修复酶体内外活性良好。5、R271G和E323A点突变的南极衣藻CPD光修复酶活性降低，M395I点突变的南极衣藻CPD光修复酶基本上失去活性。6、南极衣藻CPD光修复酶对紫外线照射后HCEC细胞的增殖和迁移都有促进作用，对紫外线照射后小鼠角膜上皮和皮肤紫外损伤修复有显著作用。