利用CRISPR/Cas9技术创制高番茄红素番茄新材料  

Creating High Lycopene Fruit Using CRISPR/Cas9 Technology in Tomato

作　　者：杨亮[1,2,3,4] 刘欢 马燕勤[1,2,3,4] 李菊 王海娥 周玉洁[1,3,4] 龙海成 苗明军[1,2,3,4] 李志 常伟 YANG Liang;LIU Huan;MA Yanqin;LI Ju;WANG Hai’e;ZHOU Yujie;LONG Haicheng;MIAO Mingjun;LI Zhi;CHANG Wei(Horticulture Research Institute,Sichuan Academy of Agricultural Sciences,Chengdu 610066,China;Sichuan Province Engineering Technology Research Center of Vegetables,Pengzhou,Sichuan 611934,China;Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province,Chengdu 610066,China;Key Laboratory of Horticultural Crops Biology and Germplasm Enhancement in Southwest Regions,Ministry of Agriculture and Rural Affairs,Chengdu 610066,China;Sichuan Institute of Edible Fungi,Chengdu 610066,China)

机构地区：[1]四川省农业科学院园艺研究所,成都610066 [2]四川省蔬菜工程技术研究中心,四川彭州611934 [3]蔬菜种质与品种创新四川省重点实验室,成都610066 [4]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 [5]四川省食用菌研究所,成都610066

出　　处：《园艺学报》2024年第2期253-265,共13页Acta Horticulturae Sinica

基　　金：国家重点研发计划项目(2022YFD1100205);四川省科技计划项目(2021YFYZ0022,2023NSFSC0162);现代农业产业技术体系建设专项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)。

摘　　要：以大果型栽培番茄资源“T048”为材料,利用CRISPR/Cas9技术对滞绿基因SlSGR1进行定向编辑。对19株转基因阳性株系进行靶位点序列分析发现,共有10株发生了编辑事件,编辑效率为52.6%。通过测序分析发现,编辑类型涉及碱基的缺失、插入及替换,多数发生在PAM序列上游3~4 bp碱基处,同时也发现了两个编辑位点间共565 bp的序列倒位。表型分析发现,T1代纯合编辑株系叶片衰老减缓,果实表现为铁锈色,且番茄红素、叶绿素及β–胡萝卜素含量显著提高。对类胡萝卜素合成关键基因进行分析发现,纯合编辑株系果实中的SGR1表达量显著降低,而PSY1表达量显著提高。结果表明,通过CRISPR/Cas9技术对滞绿基因SlSGR1进行定向编辑可有效提升番茄果实中番茄红素、β–胡萝卜素等类胡萝卜素含量,进而为番茄营养品质育种提供新种质。The large-fruited cultivated tomato resource“T048”was used as the material for targeted editing of the tomato stay-green gene SlSGR1 using CRISPR/Cas9 technology.Sequence analysis of the target sites of 19 positive transgenic plants revealed that a total of ten plants had editing events,with an editing efficiency of 52.6%.The types of editing events involved deletions,insertions and substitutions of bases,most of which occurred 3–4 bp upstream of the PAM sequence.A total of 565 bp of sequence inversion between the two editing sites was also detected.Phenotypic analysis revealed that the homozygous edited plants in the T1 generation showed slower senescence of the leaves and rusty red color fruits,with significantly higher content of lycopene,chlorophyll andβ-carotenoid,compared with unedited fruits.Analysis of the key genes for carotenoid synthesis revealed that the expression of the SGR1 in the fruit of the homozygous edited plants was significantly lower than that of the unedited plants,whereas the expression of the PSY1 was significantly higher than that of the unedited one.This study demonstrated that targeted editing of the SlSGR1 gene by CRISPR/Cas9 technology can effectively enhance the content of carotenoids such as lycopene andβ-carotene in tomato fruits,and thus provide new germplasm for tomato nutritional quality breeding.

关 键 词：番茄 基因编辑 番茄红素 类胡萝卜素 SlSGR1 

分 类 号：S641.2[农业科学—蔬菜学]