基于大肠杆菌的重离子辐射靶点序列定位和LacI突变分析的实验体系构建及验证  

Construction and Validation of Experimental System for Heavy ion Radiation Targeted Loci Localization and lacI Mutation Analysis Based on Escherichia Coli

作　　者：金铭 王婷 刘斌美 卞坡 Ming JIN;Ting WANG;Binmei LIU;Po BIAN(Institute of Intelligent Machines,Hefei Institutes of Physical Science,Chinese Academy of Sciences,Hefei 230031,China;University of Science and Technology of China,Hefei 230027,China)

机构地区：[1]中国科学院合肥物质科学研究院智能机械研究所,合肥230031 [2]中国科学技术大学,合肥230027

出　　处：《原子核物理评论》2023年第1期113-120,共8页Nuclear Physics Review

基　　金：国家自然科学基金资助项目(12135016)。

摘　　要：重离子辐射具有独特的物理和生物学特性,在诱变育种等领域有着广泛的应用,但其诱变的机制并不完全清楚。不同于传统的X和γ射线,重离子辐射具有较高的线传能密度(Linear energy transfer,LET),主要诱导团簇状的DNA损伤,其演化为遗传变异的过程更为复杂,突变类型也更难预测。目前的实验技术很难在序列水平对重离子击中DNA的靶点进行定位,这致使重离子辐射诱变机制的研究相对滞后。针对这一问题,根据重离子辐射诱导的团簇损伤核心区域富含DNA双链断裂(Double-strand break,DSB)以及同源重组机制对DSB特异性响应的特性,首先构建了四环素抗性基因(TetA)同源重组元件用于确定DNA团簇损伤的序列定位,并在重组原件侧翼连接反向突变筛选基因LacI用于团簇损伤—突变的检测,最后把该质粒转化到大肠杆菌E.coli。在此基础上,比较分析γ射线与碳重离子(80 MeV/u)辐照后同源重组和报告基因突变的情况,验证了该体系用于重离子辐射靶点序列定位及突变检测的可行性,为进一步研究重离子辐射诱变的相关机制奠定了方法学基础。Heavy ion irradiation has unique physical and biological characteristics and has been widely applied in crop and bacteria mutation breeding.However,the mechanism underlying heavy ion irradiation mutagenesis is not completely clear.Unlike the conventional X andγray,heavy ion irradiation has a high linear energy transfer(LET)and mainly induces clustered DNA damage,the evolution of which to genetic variation is more complex and mutation types are more difficult to predict.Due to the limitation of experimental techniques,it is difficult to localize the DNA target of heavy ions at the level of DNA sequence.To address this issue,we first constructed a homologous recombination(HR)element of tetracycline resistance gene(TetA)in light of high abundance of DNA double strand breaks(DSB)in the core of heavy ion radiation-induced cluster damage.And the DSB specific response of HR.We then linked a reverse mutation screening gene lacI to the TetA recombination elements to detect cluster damage-derived mutations.Finally,this plasmid was transformed into E.coli.Based on this experimental system we comparatively analyzed TetA recombinations and mutations of LacI gene after irradiation withγ-ray and carbon heavy ions(80 MeV/u).We preliminarily verified the feasibility of this research strategy and provided a methodological foundation for further investigation of the mechanisms underlying heavy ion radiation mutagenesis.

关 键 词：重离子 γ 射线 同源重组 团簇损伤 

分 类 号：Q691[生物学—生物物理学]