作物学报2024，Vol.50Issue(8)：1961-1970,10.DOI:10.3724/SP.J.1006.2024.33050
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
摘要
Abstract
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 without transgenic components,which has the potential of large-scale kernels screening with low cost and high identification efficiency.This study laid a material foundation and efficient technical basis for identifying the functions of ZmCCT10,ZmCCT9,and ZmGhd7 and creating photoperiod insensitive materials in maize.关键词
CRISPR/Cas9技术/DsRed荧光/ZmCCT10、ZmCCT9、ZmGhd7基因/玉米Key words
CRISPR/Cas9 technology/DsRed fluorescence/ZmCCT10,ZmCCT9,and ZmGhd7 genes/maize引用本文复制引用
曹晓晴,祁显涛,刘昌林,谢传晓..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[J].作物学报,2024,50(8):1961-1970,10.基金项目
本研究由北京市科技计划项目(D171100007717001)资助.This study was supported by the Beijing Science and Technology Project(D171100007717001). （D171100007717001）
