木薯MeVINV1基因的CRISPR/Cas9基因编辑载体构建及验证

Construction and Verification of CRISPR/Cas9 Gene Editing Vector of Cassava MeVINV1 Gene

液泡转化酶(vacuolar invertase,VINV)是参与植物蔗糖代谢、器官发育的关键酶之一。木薯MeVINV1在块根发育及淀粉积累阶段的表达活跃,对该基因进行编辑敲除,将有助于解析MeVINV1在木薯块根淀粉积累过程中的作用。本研究构建了MeVINV1的CRISPR/Cas9基因编辑载体,转化木薯脆性胚性愈伤组织以验证载体的编辑效果。结果发现:转化后的样品中,MeVINV1基因靶点位置出现测序多峰,而未转化样品中没有多峰出现,表明本研究构建的基因编辑载体可对MeVINV1基因进行编辑;检测分析潜在脱靶位点的序列,发现未出现脱靶现象。该研究为获得MeVINV1基因的突变体,明确其在木薯块根发育及淀粉积累过程中的功能奠定了基础。

Vacuolar invertase(VINV)is one of the key enzymes involved in sucrose metabolism and organ development in plants.The expression of cassava MeVINV1 is active at the stage of tuber development and starch accumulation.Knockout of the gene will help to analyze the role of MeVINV1 in the process of cassava root starch accumulation.In this study,the CRISPR/Cas9 gene editing vector of MeVINV1 was constructed and transformed into fragile embryogenic calli of cassava to verify the editing effect of the vector.The results showed that there were multiple sequencing peaks of MeVINV1 gene in the transformed samples,but there were no multiple peaks in the untransformed samples,which indicated that the gene editing vector constructed in this study could edit the MeVINV1 gene.Analyzing the sequence of potential off target sites,it was found that there was no off target phenomenon.This study laid a foundation for obtaining mutants of MeVINV1 gene and clarifying its function in cassava root development and starch accumulation.