基于同源重组的重离子辐射拟南芥靶点序列定位方法及可行性研究

Feasibility Study of Localizing Heavy Ion Radiation-targeted Sequences in Arabidopsis thaliana Using a Homologous Recombination-based Approach

作为粮食种质资源创新的重要途径,重离子辐射诱变技术受到了广泛关注。为了促进重离子辐射在育种上的应用,有必要研究重离子辐射在植物中诱导DNA团簇损伤的修复和突变演化机制,而在基因序列水平定位重离子的辐射靶点是该机制解析的基础。目前,尽管已经建立了一些基于常规报告基因的重离子辐射靶点定位方法,但其效率并不理想。本工作基于同源重组机制对DNA双链断裂的特异响应,以及重离子辐射靶点核心区域富含DSBs的特性,提出了一种基于同源重组报告基因的重离子辐射靶点定位新方法,通过比较γ和碳离子辐射的诱变分布特征,初步确定了该定位方法的可行性。在此基础上,在该HR-GUS报告体系侧翼增加了lacI筛选基因,进一步提高了定位效率,并揭示了重离子辐射诱变导致片段缺失和多位点突变的特性。该体系的建成为进一步阐明重离子诱变机制提供了良好的技术支撑。

Heavy-ion irradiation mutation breeding is a promising approach for food germplasm innovation and has attracted widespread attention and application.To enhance the breeding applications of heavy ion radiation,it is necessary to further investigate the mechanisms of repair and mutation evolution of DNA clustered damage induced by heavy ion irradiation in plants,based on the ability to localize the heavy-ion irradiation targets at the DNA sequence level.Currently,although some conventional reporter gene-based methods have been established to localize heavy ion radiation targets,their efficiency remains suboptimal.In this work,we propose a new method to accurately localize the targeted loci of heavy-ion irradiation on DNA molecules in Arabidopsis thaliana,based on the abundance of DNA double-strand breaks(DSB) in the core of clustered DNA damage and the specific response of the homologous recombination mechanism to DSB,and its feasibility was initially determined by comparing the mutagenic distribution characteristics of γ and carbon ion radiation.To further improve the localization efficiency,by linking the lacI screening gene to the flanking sequence of the HR-GUS gene,we found that the characteristics of heavy-ion irradiation mutagenesis were fragment deletion and multi-loci mutation.This experimental system will provide a good technical support for further elucidating the mechanisms of heavy ion mutagenesis.