利用CRISPR-Cas9技术快速创制番茄雄性不育系  

Rapid Breeding of Tomato Male Sterile Lines by CRISPR-Cas9 Technology

作　　者：杨亮[1] 刘欢[1] 李菊[1] 李志[1] 苗明军[1] 龙海成 周玉洁 王海娥[1] 常伟[2] Yang Liang;Liu Huan;Li Ju;Li Zhi;Miao Mingjun;Long Haicheng;Zhou Yujie;Wang Haie;Chang Wei(Horticultural Crops Biology and Germplasm Enhancement in Southwest Regions Key Laboratory of Ministry of Agriculture and Rural Affairs,Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province,Horticulture Research Institute,Sichuan Academy of Agricultural Sciences,Chengdu,610066;Institute of Agricultural Resources and Environment,Sichuan Academy of Agricultural Sciences,Chengdu,610066;College of Resources,Sichuan Agricultural University,Chengdu,611130)

机构地区：[1]四川省农业科学院园艺研究所,蔬菜种质与品种创新四川省重点实验室,农业农村部西南地区园艺作物生物学与种质创制重点实验室,成都610066 [2]四川省农业科学院农业资源与环境研究所,成都610066 [3]四川农业大学资源学院,成都611130

出　　处：《分子植物育种》2023年第11期3619-3627,共9页Molecular Plant Breeding

基　　金：四川省科技计划项目(2021YFYZ0022,2023NSFSC0162);国家现代农业产业技术体系四川省蔬菜创新团队项目[川农函(2019)472号];四川省农业科学院“1+9”揭榜挂帅科技攻关项目(1+9KJGG001)共同资助。

摘　　要：为创制番茄雄性不育系,利用CRISPR-Cas9技术对番茄雄性不育基因SlTM6进行定向编辑,在SlTM6基因第一外显子区域设计两个靶位点,构建CRISPR-Cas9基因敲除载体,并通过农杆菌转化番茄优异自交系T048。对8株转基因阳性株系进行靶位点序列分析发现,共有6株发生了编辑事件,编辑效率为75%,其中,双等位编辑株系表现为柱头外露,雄蕊发育不完全且无花粉活性,而杂合编辑株系可育。对T0代杂合编辑株系进行继代培养后获得无外源载体成分的纯合雄性不育株系,同时开发分子标记并进行验证。本研究表明,通过CRISPR-Cas9技术对雄性不育基因SlTM6进行定向编辑可快速创制雄性不育番茄新种质。In order to create tomato male sterile lines,CRISPR-Cas9 technology was used to edit tomato male sterile gene SlTM6 directionally.Two targets were designed in the first exon region of SlTM6 and integrated into CRISPR-Cas9 vector,then the constructed vector was transformed into the tomato excellent inbred line T048 through Agrobacterium tumefaciens.According to the target sequences of 8 transgenic positive lines,a total of 6 plants were edited with an editing efficiency of 75%.Among them,the biallelic editing lines showed stigma expo-sure,incomplete stamen development and no pollen activity,while the heterozygous editing lines were fertile.Af-ter subculture of To generation heterozygous edited lines,homozygous male sterile lines without exogenous vector components were obtained.At the same time,molecular markers were developed and validated according to the mutated sequence.This study showed that CRISPR-Cas9 technology can be used to edit SITM6 to rapidly create new male sterile germplasm of tomato.

关 键 词：番茄(Solanum lycopersicum) 雄性不育 SlTM6 CRISPR-Cas9 基因组编辑 

分 类 号：S641.2[农业科学—蔬菜学]