CRISPR/Cas9技术介导烟草CENH3基因突变体的获得  

CRISPR/Cas9 mediated acquisition of tobacco CENH3 gene mutants

作　　者：韩平安 孙瑞芬 常悦 唐宽刚 王良 聂利珍 张自强 梁亚晖 吴新荣 李晓东 HAN Ping′an;SUN Ruifen;CHANG Yue;TANG Kuangang;WANG Liang;NIE Lizhen;ZHANG Ziqiang;LIANG Yahui;WU Xinrong;LI Xiaodong(Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot 010031,China;Inner Mongolia Key Laboratory of Sugarbeet Genetics and Germplasm Enhancement,Hohhot 010031,China)

机构地区：[1]内蒙古自治区农牧业科学院,内蒙古呼和浩特010031 [2]内蒙古自治区甜菜品种遗传改良与种质创制重点实验室,内蒙古呼和浩特010031

出　　处：《北方农业学报》2022年第3期1-8,共8页Journal of Northern Agriculture

基　　金：国家自然科学基金地区基金项目(31860407);内蒙古自治区科技重大专项项目(ZDZX2018015);国家糖料产业技术体系项目(CARS-170104);内蒙古自治区“草原英才”创新人才团队项目(甜菜生物技术产业创新团队);内蒙古农牧业创新基金项目(2022CXJJN09)。

摘　　要：【目的】通过CRISPR/Cas9技术对烟草CENH3基因进行编辑,获得CENH3基因发生突变的植株,确定编辑载体的编辑效率与突变类型,建立高效基因组编辑技术体系。【方法】根据CENH3序列设计特异性编辑位点,构建CENH3编辑载体;采用农杆菌介导法转化烟草获得转基因植株,通过测序明确转基因植株的目标基因编辑效率与CENH3突变类型;利用微滴式数字PCR技术筛选低拷贝突变体植株。【结果】随机检测的11株转基因烟草植株,有6株被编辑,编辑效率为54.5%;编辑植株中,靶标1未发生突变,靶标2发生4种突变类型。6株编辑植株均为低拷贝植株,拷贝数为0.3~1.2。【结论】实现了对烟草CENH3的定点编辑,建立了CRISPR/Cas9介导的烟草CENH3基因组编辑体系。【Objective】Using CRISPR/Cas9 technology,the tobacco CENH3 gene was edited,and mutant plants with CENH3 gene mutations were obtained.An effective genome editing technology system was established after the editing efficiency and mutation type of the editing vector were identified.【Methods】The CENH3 editing vector was designed and constructed according to tobacco CENH3 gene sequence.Through Agrobacterium-mediated transformation,transgenic tobacco plants were produced,and sequencing was used to identify the CENH3 mutation types and the target gene editing efficiency of the transgenic plants.Droplet digital PCR was used to screen low-copy mutant plants.【Results】Among the 11 transgenic tobacco plants randomly tested,6 were edited,and the editing efficiency was 54.5%.In the edited plants,target 1 was not edited,and target 2 had four mutation types.All six of the edited plants had low copy numbers,ranging from 0.3 to 1.2.【Conclusion】The fixed-point editing of tobacco CENH3 was successfully achieved and CRISPR/Cas9 mediated tobacco CENH3 genome editing system was established.

关 键 词：烟草 CENH3基因 CRISPR/Cas9 转基因 基因组编辑 

分 类 号：Q943.2[生物学—植物学]