编辑ZmCCT10、ZmCCT9、ZmGhd7基因的串联DsRed荧光表达盒的CRISPR/Cas9系统的构建及验证  

Construction and verification of the CRISPR/Cas9 system containing DsRed fluorescent expression cassette for editing of ZmCCT10,ZmCCT9,and ZmGhd7 genes in maize

作　　者：曹晓晴 祁显涛 刘昌林[1] 谢传晓[1] CAO Xiao-Qing;QI Xian-Tao;LIU Chang-Lin;XIE Chuan-Xiao(Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]中国农业科学院作物科学研究所,北京100081

出　　处：《作物学报》2024年第8期1961-1970,共10页Acta Agronomica Sinica

基　　金：北京市科技计划项目(D171100007717001)资助。

摘　　要：CCT家族基因影响植物开花,在玉米中,ZmCCT10、ZmCCT9基因是光周期敏感基因,ZmGhd7基因是与开花期相关的基因。利用CRISPR/Cas9技术靶向编辑ZmCCT10、ZmCCT9、ZmGhd7基因为研究基因的功能和快速改良玉米的开花期提供了可能。本研究以玉米ZmCCT10、ZmCCT9、ZmGhd7基因为编辑对象,以KN5585为稳定转化受体、以CML312SR、LCL-1、LCL-2为预改良的晚熟材料受体,首先通过Sanger测序验证了3个基因靶标区域在4份玉米材料中的保守性,其次根据sgRNA设计原则选择了1个sgRNA复合编辑3个基因,并利用同源重组方法构建了将由胚特异性启动子Zm3896驱动的DsRed表达盒和由ZmU6-2启动子驱动的sgRNA表达盒串联的CRISPR/Cas9基因编辑敲除载体CCT-CPD,然后采用酶切法和Sanger测序法分析T0代KN5585中3个基因的突变率和突变类型,验证了该系统的基因编辑效果,最后通过对稳定遗传转化植株所结籽粒在籽粒水平、组织水平进行DsRed荧光标记表型鉴定,验证了该系统中DsRed荧光筛选标记的有效性。在此基础上,通过杂交育种法以晚熟材料为母本、以T1代KN5585阳性株为父本获得F1并经过DsRed荧光筛选获得含有有效编辑转基因元件的晚熟材料。本研究构建的编辑ZmCCT10/ZmCCT9/ZmGhd7基因的串联DsRed荧光表达盒的CRISPR/Cas9系统为创制单基因突变体,双基因突变体,三基因突变体奠定了基础,该系统中DsRed荧光筛选标记的应用可以快速筛选区分有无转基因成分的玉米籽粒,成本低,鉴定效率高,具有大规模籽粒筛选的潜力,本研究为鉴定ZmCCT10、ZmCCT9、ZmGhd7三个基因的功能和创制玉米光周期钝感材料奠定了材料基础和高效的技术基础。The CCT family genes affect plant flowering time.In maize,ZmCCT10 and ZmCCT9 are photoperiod sensitive genes,and ZmGhd7 is a gene related to the flowering time.Targeted editing of ZmCCT10,ZmCCT9,and ZmGhd7 genes using CRISPR/Cas9 technology provides the possibility to study the function of three genes and to rapidly improve the flowering time of maize.In this study,maize ZmCCT10,ZmCCT9,and ZmGhd7 were used as editing objects.The inbred line KN5585 was used as a stable transforming receptor,and CML312SR,LCL-1,and LCL-2 were used as pre-modified late-flowering lines.Firstly,the conservation of the target regions of the three genes in four maize lines was verified by Sanger sequencing.Secondly,one sgRNA was selected to co-edit three genes based on sgRNA design principles.The CRISPR/Cas9 gene editing knockout vector CCT-CPD was constructed using homologous recombination,which contained the DsRed expression cassette driven by embryo-specific promoter Zm3896 and the sgRNA expression cassette driven by the ZmU6-2 promoter.Next,the mutation rate and mutation type of the three genes in T0 generation KN5585 were analyzed by enzyme digestion method and Sanger sequencing,and the gene editing effect of the CRISPR/Cas9 system was verified.Finally,the seeds produced by stable genetic transformation plants were verified by the DsRed fluorescent labeling phenotype at the kernel level and tissue level.On this basis,F1 was obtained by cross breeding using late flowering lines as female parent and T1 generation KN5585 positive plant as male parent,and late flowering lines containing effective edited transgenic elements were obtained by DsRed fluorescence screening.The CRISPR/Cas9 system for editing ZmCCT10,ZmCCT9,and ZmGhd7 genes containing DsRed fluorescent expression cassette constructed,in this study,laid a foundation for the creation of single-gene mutants,double-gene mutants,and/or triple-gene mutants.The application of DsRed fluorescent screening markers in this system can quickly screen and distinguish corn kernels with or wi

关 键 词：CRISPR/Cas9技术 DsRed荧光 ZmCCT10、ZmCCT9、ZmGhd7基因 玉米 

分 类 号：S513[农业科学—作物学] Q943.2[生物学—植物学]