【作者】 刘敏；

【导师】 曹毅；

【作者基本信息】 四川大学 ， 微生物学， 2003， 硕士

【摘要】 紫外光照射可导致生物体产生两种损伤：(6-4)光产物和环丁烷嘧啶二聚体。针对这些嘧啶二聚体的损伤，许多生物体可产生一种名为光裂合酶的蛋白，这种蛋白吸收UV-A／蓝光，通过一个光依赖的作用机制(光复活作用)来分解这些光产物，完成修复功能。这些光裂合酶分别作用(6-4)光产物和环丁烷嘧啶二聚体，因此，该酶可分为(6-4)光裂合酶和环丁烷嘧啶二聚体光裂合酶。目前已从高等生物拟南芥、鲐类、果蝇、人类和非洲爪蟾蜍属中克隆到有(6-4)光裂合酶活性的基因，本研究从盐生杜氏藻Dunaliella salina中克隆到(6-4)光裂合酶的基因，并将该基因在大肠杆菌中得以表达，这是首次在藻类中克隆到(6-4)光裂合酶基因，对光裂合酶的研究具有重要意义。研究的主要内容包括： 1．运用BLAST方法对EST序列进行分析，初步预测该EST序列为光裂合酶／蓝光受体蛋白质家族中一个成员的部分cDNA。 2．利用3’RACE法扩增此cDNA序列的3’端部分，所得片段长度为2575bp，含一个完整的开放读框，长1800bp，可编码600个氨基酸；利用生物信息学法对此扩增序列进行分析，包括同源性分析、序列的读框分析、蛋白质的保守性分析以及该蛋白的进化关系分析，预测出此序列编码了D. salina的(6-4)光裂合酶。 3．克隆编码D. salina的(6-4)光裂合酶的开放阅读框序列，并以BamHI和XhoI位点定向插入到原核表达载体pGEX-4T-1中，构建成pGEX-4T-1／6-4载体，并将此载体转化到大肠杆菌J-M109中。 4.用工PTG诱导含pGEX一4T一1/6一4的转化菌，提取初提物中的总蛋白，进行SDS一聚丙烯酞胺凝胶电泳，检测表达的融合蛋白大小越为gokD。 5.工PTG诱导过的转化菌，经过30秒的紫外光照射后，分别进行光培养或暗培养，计算存活率，从而证明了及sa了z’na的(6一4)光裂合酶在大肠杆菌中可进行光修复活性作用。更多还原

【Abstract】 UV radiation induced two major classes of pyrimidine dimmers:the pyrimidine (6-4) pyrimidone photoproduct (6-4 product) and the cyclobutane pyrimidine dimmer(CPD). Many organisms produce enzymes, termed photolyases, that specifically bind to these damage products and split the via a UV-A/blue light-dependent mechanism(photoreactivation), thereby reversing the damage. These two photolyase are specific for either CPDs(CPD photolyase) or 6-4 products (6-4 photolyase). A gene that expresses a protein with 6-4 photolyase activity in vitro, was recently cloned from high organisms(Arabidopsis thaliam , Drosophila melanogaster , Danio rerio , Xenopus laevis and Homo sapiens).We report here the cloning of a homolog of this gene from Dunaliella salina . This cloned gene produces as protein with photolyase activity when expressd in Escherichia coli. It’ s the first cDNA code (6-4)photolyase found in low orgnism(alga), and this study is important for the reseach of 6-4 photolyase. The methods and analysis as bellows:1. This EST was analyzed by method of BLAST, and the conclusion suggests that the sequence probably be a partial cDNA that can code one of protein family of photolyase/ blue light photoreceptor.2. Amplify the 3’ sequence of EST by 3’ -RACE, the result is 2575bp , and the open reading frame (ORF) is 18OObp, coding 600 AA , thenanalyze this 3’ UTR by bioinfomatice methods, including BLAST, ORF analysis, conservative analysis and phylogenetic analysis. The sequence probably code a (6-4) phtolyase.3. Vector pGEX-4T-l/6-4 was constructed by inserting the BamHI/XhoI ORF sequence of (6-4) phtolyase into the vector pGEX-4T-l. The E. coli strain JM109 was transformed with resultant plasmid pGEX-4T-1/6-4.4. The transformation was induced with IPTG, then the total protein from cell extract was analyzed by electrophoresis on a 8% SDS-PAGE in order to validate the GST fusion protein, and the fusion protein is about 90kD.5. After induced with IPTG, JM109 with pGEX-4T-l/6-4 was radiated by UV for 30 seconds, cultivate these E. coli with light or without light. The survival rate proved the gene of D. salina (6-4) photolyase has photoreactivation.更多还原