利用CRISPR/Cas9技术创制黄果番茄新种质  

Creating New Germplasm of Yellow Fruit Tomato(Solanum lycopersicum)Using CRISPR/Cas9 Technology

作　　者：龙海成 马燕勤 周玉洁 常伟 李菊[1,2] 李志 苗明军[1,2] 杨亮 LONG Hai-Cheng;MAYan-Qin;ZHOU Yu-Jie;CHANGWei;LI Ju;LI Zhi;MIAO Ming-Jun;YANG Liang(Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province/MOA of Key Laboratory of Horticultural Crops Biology and Germplasm Enhancement in Southwest Regions,Horticulture Research Institute,Sichuan Academy of Agricultural Sciences,Chengdu 610066,China;Sichuan Province Engineering Technology Research Center of Vegetables,Pengzhou,Sichuan 611934,China;Sichuan Institute of Edible Fungi,Chengdu 610066,China;College of Resources,Sichuan Agricultural University,Chengdu 611130,China)

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]四川省蔬菜工程技术研究中心,彭州611934 [3]四川省食用菌研究所,成都610066 [4]四川农业大学资源学院,成都611130

出　　处：《农业生物技术学报》2024年第7期1693-1702,共10页Journal of Agricultural Biotechnology

基　　金：国家现代农业产业技术体系四川省蔬菜创新团队(蔬菜分子辅助育种岗位)项目[川农函(2019)472号];四川省科技计划项目(2021YFYZ0022,2023NSFSC0162);四川省财政自主创新专项项目(2022ZZCX050)。

摘　　要：目前国内市场上优异黄果番茄(Solanum lycopersicum)品种相对稀缺,为了满足市场需求,提高种业竞争力,本研究以'MoneyMaker'('MM')番茄为材料,利用CRISPR/Cas9技术,对番茄八氢番茄红素合成酶1(phytoene synthase 1,SlPSY1)基因(Solyc03g031860)进行定向基因编辑来创制黄色果实番茄。选择SlPSY1基因外显子区域2个序列特异的sgRNA作为靶位点,构建CRISPR/Cas9双元表达载体p KSE401,通过根癌农杆菌(Agrobacterium tumefaciens)介导的叶盘转化法对'MM'番茄材料进行遗传转化。结果显示,16株再生苗中鉴定出了12株阳性转基因植株,阳性转化率为75%;对阳性转基因植株的靶位点测序分析发现,有8株在2个靶位点上共发生21次编辑事件,基因编辑效率为66.7%,且均未发生脱靶现象;获得的突变植株中有5株在成熟期果实表现为黄色。本研究利用CRISPR/Cas9技术在'MM'番茄上成功创制了SlPSY1基因编辑材料,为今后快速创制番茄黄果资源提供技术及材料基础。At present,excellent yellow fruit tomato(Solanum lycopersicum)varieties are relatively scarce in the domestic market.In order to meet the market demand and improve the competitiveness of the seed industry,the tomato variety'MoneyMaker'('MM')was used to create yellow fruit by targeted gene editing of the tomato phytoene synthase 1(SlPSY1)using CRISPR/Cas9 technology.Two sequences specific sgRNAs in the exon region of the SlPSY1 were selected as target sites to construct the CRISPR/Cas9 binary expression vector pKSE401.The'MM'tomato material was genetically transformed using Agrobacterium tumefaciens mediated leaf disc transformation method.The results showed that 12 transgenic-positive plants were detected in 16 regenerated seedlings,with a positive transformation rate of 75%;Sequencing analysis of the target sites of the positive transgenic plants showed that 8 plants had a total of 21 editing events at 2 target sites,with a gene editing efficiency of 66.7%,and none of them was off-target;Five of the edited plants showed yellow fruit phenotype at maturity.In this study,CRISPR/Cas9 technology was used to successfully created SlPSY1 gene editing plants in'MM'tomato,which will provide a technical and material basis for the rapid creation of tomato yellow fruit resources in the future.

关 键 词：番茄 CRISPR/Cas9 八氢番茄红素合成酶1(PSY1) 基因编辑 黄果番茄 

分 类 号：S641.2[农业科学—蔬菜学]