Rolloos Martijn, Hooykaas Paul J J, van der Zaal Bert J
Department of Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden, Sylviusweg 72, 2333 BE, The Netherlands.
Sci Rep. 2015 Feb 9;5:8345. doi: 10.1038/srep08345.
Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.
农杆菌介导的转化（AMT）已被生物技术学家视为引入或改变植物遗传特性的首选技术。然而，在植物中，除非能够在感兴趣的位点在DNA中产生双链断裂（DSB），否则几乎不可能预先确定转移的T链的整合位点。在本研究中，我们使用模式生物酿酒酵母来研究是否可以通过IV型分泌系统（T4SS）伴随T-DNA转移实现农杆菌介导的位点特异性内切酶的转位，以及这是否可以提高基因靶向效率。除此之外，还研究了不同染色质状态对靶向整合的影响。结果发现，农杆菌介导的归巢内切酶I-SceI的转位对通过同源修复（HR）途径的T-DNA整合具有积极作用。此外，我们获得的证据表明，核小体的去除对I-SceI促进的HR介导的T-DNA整合具有积极作用。相反，在整合位点诱导核小体形成会消除转位的I-SceI对T-DNA整合的积极作用。
