摘要
CRISPR/Cas9是实现基因精准突变的有效工具而被广泛应用到功能基因组学研究中。通常需要在已编辑后代的基因组中剔除此编辑体系,以防Cas9进一步编辑导致复杂的脱靶效应。本研究利用可视化的CRISPR/Cas9基因编辑系统敲除拟南芥AtPAE4基因,测序结果显示AtPAE4在第一个外显子的第478号碱基前面插入了一个碱基A,导致氨基酸的移码突变而提前终止。利用种皮特异启动子控制表达的m Cherry荧光蛋白,可以在T_(1)代纯合突变体种子群体中分别鉴定出发荧光和不发荧光的种子家系,抗性筛选及分子检测发现不发荧光家系为Cas9-free的AtPAE4突变家系。对AtPAE4突变体的根、茎、叶进行qPCR分析发现AtPAE4在根、茎、叶上的转录水平均显著下调。这些结果表明可视化CRISPR/Cas9系统在基因编辑后代中可快速鉴定到Cas9-free突变体植株,这有助于为进一步研究靶基因功能而获得背景清晰的编辑材料。
CRISPR/Ca s9,an effective tool for precise gene editing,is now widely used in functional genomic studies.Usually,the editing system is required to be eliminated in edited offsprings to prevent genetic complication induced by continuing presence of CRISPR/Cas9.In this study,the AtPAE4 gene was targeted by using a visual CRISPR/Cas9 system.Sanger sequencing indicated that a base A was inserted at the 478 thposition of the first exon,suggesting a frameshift mutation and early termination of translation in AtPAE4 gene.Based on the expression of mCherry fluorescent gene under the control of seed-expressing promoter At2 S3,the offsprings of T_(1) homozygous mutation generation were separated into fluorescence-positive and fluorescence-negative groups.Further negative selection and PCR examination indicated that fluorescence-negative plants were AtPAE4 Cas9-free mutants.Quantitative PCR analysis showed that the transcripts of AtPAE4 gene were significantly down-regulated in roots,stems and leaves compared to the wild type.These results indicated that the visual CRISPR/Cas9 construct can be rapidly segregated out in the offsprings of CRISPR transgenic lines,facilitating the identification of genetically edited material with clearer background.
作者
王媛媛
金天麟
刘小芳
蒋梦婷
梁大成
Wang Yuanyuan;Jin Tianlin;Liu Xiaofang;Jiang Mengting;Liang Dacheng(Hubei Collaborative Innovation Center for Grain Industry,Yangtze University,Jingzhou,434025;Hubei Key Laboratory of Waterlogging Disaster and Wetland Agriculture,Engineering Research Center of Ecology and Agricultural Use of Wetland,Ministry of Education,Yangtze University,Jingzhou,434025)
出处
《分子植物育种》
CAS
北大核心
2021年第11期3605-3611,共7页
Molecular Plant Breeding
基金
国家自然科学基金项目(31671257)资助。
