基于CRISPR-Cas9介导敲除盘基网柄菌erkA基因的方法建立和erkA趋电性研究

Establishment of erkA Gene Knockout System in Dictyostelium discoideum Based on CRISPR/Cas9 and Exploration of erkA in Electrotaxis

以盘基网柄菌为研究对象,首先建立基于成簇的规律间隔短回文重复序列及其相关蛋白9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9,CRISPR/Cas9)基因编辑系统的erkA基因敲除方法,DNA测序和蛋白质印迹实验都证明所设计靶点都能有效地编辑erkA;随后对erkA突变株的趋电性进行探究,发现在12 V/cm的直流电场中,erkA突变株仍维持着与野生型细胞接近的趋电性,仅在运动速度上表现出增加趋势.在盘基网柄菌中利用CRISPR/Cas9系统成功构建了基因突变株,也明确了在盘基网柄菌中erkA基因并不是细胞感应电场的关键基因.

In this study,the model organisms Dictyostelium discoideum was taken as research object to uncover the role of erkA in electrotaxis.Firstly,the erkA gene knockout method based on CRISPR/Cas9 gene editing system is established.DNA sequencing and Western blotting experiments have proved that the designed targets can effectively edit erkA.Secondly,the electrotaxis of the erkA mutant was investigated.The results showed that erkA mutant still maintained electrotaxis close to the wild-type cells in a DC electric field of 12 V/cm,and only showed an increasing trend in the movement speed.The results of this study provide a reference for the construction of gene mutant strains using CRISPR/Cas9 system in Dictyostelium discoideum,and also clarify that erkA gene is not a key gene of electrotaxis in Dictyostelium discoideum.