摘要
CRISPR/Cas9介导的基因组编辑技术已经逐渐成熟并在多种植物中得到成功应用,促进了基因功能的研究。小麦籽粒硬度是决定小麦加工品质的重要性状,Pinb是控制籽粒硬度的关键基因之一。本研究以小麦Pinb基因为对象,联合采用常规PCR和Golden Gate cloning技术,构建了包含小麦Pinb基因启动子区双靶点的CRISPR/Cas9基因编辑载体。将重组载体转入小麦原生质体中进行sgRNA活性的检测,在两个靶点位置均发生了突变,编辑效率分别为4.57%和4.37%,基因编辑类型包括碱基替换和碱基缺失。该研究为在小麦中实现Pinb基因的定点突变奠定了基础,对其功能研究和小麦品质改良均具有重要意义。
The genome editing technology system mediated by CRISPR/Cas9 has ripen and successfully applied in many plants,which promotes the study of gene function. Hardness of wheat grain is an important trait for determining the quality of wheat processing. The Pinb gene is one of the key genes controlling grain hardness. In the study,we chose the Pinb gene of wheat as research object,and constructed a CRISPR/Cas9 genome editing vector containing dual targets in the Pinb gene promoter region of wheat by conventional PCR and Golden Gate cloning technology. Then we transferred the recom;binant vector into wheat protoplast and detected the activity of sgRNA. We found the dual targets position had mutated,and the gene editing types included base substitution and base deletion,and the corresponding editing efficiencies were 4. 57% and 4. 37%,respectively. The results laid the foundation for realizing the site-directed mutagenesis of the Pinb gene,which was of great significance for the functional study of Pinb gene and the improvement of wheat quality.
作者
张淑娟
张荣志
宋国琦
李玉莲
高洁
李玮
高世庆
李根英
Zhang Shujuan;Zhang Rongzhi;Song Guoqi;Li Yulian;Gao Jie;Li Wei;Gao Shiqing;Li Genying(Crop Research Institute,Shandong Academy of Agricultural Sciences/Key Laboratory of Wheat Biology and Genetic Breeding in the Northern of Yellow and Huai Region,Ministry of Agriculture/Wheat and Maize National Engineering Laboratory,Jinan 250100,China;Beijing Hybrid Wheat Engineering Technology Research Center,Beijing 100097,China)
出处
《山东农业科学》
2020年第1期1-9,共9页
Shandong Agricultural Sciences
基金
国家转基因生物新品种培育重大专项(2018ZX08009-10B)
山东省农业科学院农业科技创新工程项目(CXGC2019G02)
山东省农业良种工程项目(2019LZGC015)
国家重点研发计划项目“主要农作物品质性状形成的分子基础”(2016YFD0100500)
