利用DNA洗牌技术构建GRATEN体系用于基因组编辑的分析-微生物学专业论文.docxVIP

聊城大学硕士学位论文 聊城大学硕士学位论文 PAGE PAGE iii ABSTRACT Gene mutation has been the most effective way to study gene function. With the rapid development of modern genome sequencing technology, the functions of a large number of genes or regulatory factors are required to be identified. Therefore, to establish a precise and efficient technology of site-directed genomic mutagenesis, the use of reverse genetics to study gene (or DNA) function is one of the hotspots of the current functional genomics research. Artificial zinc-finger nuclease technology (ZFN), transcription activator-like effector nuclease technology (TALEN) and RNA-mediated clustered regularly interspaced short palindromic repeats (CRISPR) systems can produce DNA double-strand breaks precisely at the specific sites of the genome, which achieves mutations of gene insertions, deletions, base substitutions by intracellular endogenous DNA repair mechanisms (homologous recombination and non-homologous end joining repair). Compared with the traditional gene editing techniques, the new technology not only retains characteristics of modifications at specific sites, but also can be applied to more species. It greatly improves the efficiency of the gene designated editing. The time of constructing vector is shortened. The costs are reduced. It also provides a broad platform for the realization of the designated genome editing. However, recent studies have found that CRISPR system can generate very high rate of off-target . In this study, we cloned multiple gene fragments and achieved mutations of two amino acid on specific site at once time by DNA shuffling. We also constructed an expression vector using DNA shuffling technology and executed genome editing using GRATEN (guided RNA and tethered endonuclease) for the first time in the plant. We explored the possibility of site-directed genome mutagenesis by GRATEN system in the plant and then transiently expressed in tobacco leaves, and conducted a preliminary study of its