摘要
为了构建番茄CRISPR/Cas9介导的多基因编辑体系,用SlU6-2p、SlU6-3p、SlU6-7p、SlU3-5p、SlU3-9p和SlU6-5p启动子分别替换pKSE401和pCBC-DT1T2载体中的拟南芥U6启动子,构建了1个双元载体(p MGET)、2个中间载体(pKC-S2M和pKC-S3M)和3个gRNA模块载体(pCBC-S1、pCBC-S2和pCBC-S3)。为了测试该多基因编辑体系,通过PCR扩增、Goldengate克隆和同尾酶技术,将含有6个番茄果实性状相关基因Green flesh(GF)、Ovate(O)、Locule number(LC)、SlMYB12(Y)、Tangerine(T)、Uniformripening(U)靶点序列的sgRNA聚合构建多基因编辑载体pMGET-OYGTULC和pMGET-TULCOYG,检测6个基因同时被编辑的效率分别为44.00%和11.76%。用携带pMGET-OYGTULC载体的根癌农杆菌转化番茄材料获得2株6个基因均被编辑的植株,序列变异为单个碱基的插入、单个或多个碱基的缺失及大片段的缺失等多种形式。本研究中该多基因编辑体系可以高效地应用于番茄多基因编辑,并且可以得到稳定遗传的突变体,可为番茄基础研究和遗传改良提供一个简便的工具箱。
In order to construct a CRISPR/Cas9 system for multiplex gene editing in tomato,Arabidopsis U6 promoters in pKSE401 and pCBC-DT1T2 vectors were replaced with SlU6-2p,SlU6-3p,SlU6-7p,SlU3-5p,SlU3-9p and SlU6-5p,respectively.Accordingly,a binary vector(pMGET),two intermediate vectors(pKC-S2M and pKC-S3M),and three gRNA module vectors(pCBC-S1,pCBC-S2 and pCBC-S3)were constructed.In order to test the system for multiplex gene editing in tomato,multiplex gene editing vectors pMGET-OYGTULC and pMGET-TULCOYG were constructed by aggregating sgRNA expression cassettes containing the target sequences of six tomato fruit trait-related genes Green flesh(GF),Ovate(O),Locule number(LC),SlMYB12(Y),Tangerine(T)and Uniform ripening(U),through PCR amplification,Golden gate cloning and isocaudamer technique,and the editing efficiency of six genes at the same time of the two vectors was 44.00%and 11.76%,respectively.The tomato material was transformed by Agrobacterium tumefaciens carrying pMGET-OYGTULC vector to obtain two plants with six genes edited.The sequence variations were in the form of single base insertion,single or multiple base deletion and large fragment deletion.In this study,the CRISPR/Cas9 system can be efficiently used for multiplex gene editing in tomato,and stable genetic mutants can be obtained,which can provide a versatile toolbox for basic research and genetic improvement in tomato.
作者
杨孟霞
刘晓林
曹雪
魏凯
宁宇
杨沛
李珊珊
陈紫月
王孝宣
国艳梅
杜永臣
李君明
刘磊
李鑫
黄泽军
YANG Mengxia;LIU Xiaolin;CAO Xue;WEI Kai;NING Yu;YANG Pei;LI Shanshan;CHEN Ziyue;WANG Xiaoxuan;GUO Yanmei;DU Yongchen;LI Junming;LIU Lei;LI Xin;HUANG Zejun(State Key Laboratory of Vegetable Biobreeding,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China)
出处
《园艺学报》
CAS
CSCD
北大核心
2023年第6期1215-1229,共15页
Acta Horticulturae Sinica
基金
国家自然科学基金项目(31872949,31672154)
国家重点研发计划项目(2018YFD1000800)
国家大宗蔬菜产业技术体系建设专项资金项目(CARS-23-A06)。