利用CRISPR/APOBEC1 单碱基编辑系统创制多价除草剂抗性棉花

棉田杂草不仅与棉花争夺阳光、水份和养分，同时也是农作物病虫害的中间宿主，传播病虫害，严重威胁棉花的产量和品质。除草剂可以有效清除田间杂草，除草剂抗性棉花种植可有效降低棉田除草劳动强度。目前，抗除草剂棉花主要以导入外源抗性基因及筛选突变体获得，存在安全评价及抗性单一的问题。本课题组将Cas9缺口酶、胞嘧啶脱氨酶、尿嘧啶糖基化酶抑制剂融合构建了适用于棉花遗传转化的多位点单碱基编辑系统，通过启动子优化，避免过量表达胞嘧啶脱氨酶与DNA结合造成的脱靶现象，实现棉花棉花基因组高效的单碱基编辑，提高棉花分子育种的精准性。本项目拟通过棉花多位点单碱基编辑系统（tRNA-sgRNA-GhBE）定向突变GhEPSP、GhALS、GhAHAS以获得除草剂抗性突变材料，创制多价除草剂抗性棉花，有效防止田间抗性杂草产生，减少杂草对棉花产量及品质的影响。

Weeds in cotton fields seriously affect the yield and quality of cotton fiber, which not only compete with cotton for light, water and nutrients, but also act as intermediate hosts and refuges of many crop diseases and insect pests. Herbicides can eliminate weeds in the field effectively, and the planting herbicide-resistant cotton can effectively decrease the labour intensity of weeding in the field. At present, herbicide-resistant cotton is mainly acquired by introducing exogenous resistance genes and mutations, and there are severe concerns about GMO evaluation and weeds mutants for herbcide resistance. In this study, we synthesized Cas9 nickase (nCas9), cytosine deaminase (APOBEC1), and uracil glycosylase inhibitor (UGI) to construct a base editing system (GhBE3) for cotton base editing via Agrobacterium-mediated transformation systems. This project aims to develop herbicide-resistant cotton materials by the point mutations of cotton endogenous herbicide target genes, GhEPSP, GhALS and GhAHAS, using cotton multi-site single base editing system (tRNA-sgRNA-GhBE). In this way, we can prevent the emergence of herbicide-resistant weeds in the field and reduce the impact of weeds on cotton fiber yield and quality effectively.