作物学报2018，Vol.44Issue(2)：227-235,9.DOI:10.3724/SP.J.1006.2018.00227
基于棉花U6启动子的海岛棉CRISPR/Cas9基因组编辑体系的建立
Establishment of CRISPR/Cas9 Genome Editing System Based on GbU6 Pro-moters in Cotton (Gossypium barbadense L.)
摘要
Abstract
CRISPR/Cas9 genome editing is a powerful tool for genes functional analyses, and the mutation of endogenous genes has been successfully implemented in many organisms using the tool. Two cloned U6 promoter from sea island cotton Xinhai 16 were used to construct CRISPR/Cas9 gene editing vectors with target(GbGGB and GBERA1)DNA fragments from Xinhai 16 respectively. Through PEG method, the core fragments (including GbU6::sgRNA and CAMV35S::Cas9) of the CRISPR/Cas9 gene editing vectors enriched by PCR method were transformed into the cotton protoplast prepared from the embryo callus of Xinhai 16. The mutation of endogenous target genes was successfully detected by a restriction enzyme PCR (RE-PCR) assay of protoplast genome. The cloning and sequencing of the PCR product, showed that the two Cas9-GbU6-sgRNA vectors could both induce targeted mutagenesis. Sequence analysis revealed that most of the mutations were nucleotide substitutions and the few were nucleotide deletion. The results indicate that the CRISPR/Cas9 gene editing vector system based on GbU6 promoter can realize targeted mutagenesis in sea island cotton, which provides an important technical basis for cotton functional genomics re-search.关键词
棉花/原生质体/CRISPR/Cas9/基因组编辑Key words
cotton/protoplast/CRISPR/Cas9/genome editing引用本文复制引用
李继洋,雷建峰,代培红,姚瑞,曲延英,陈全家,李月,刘晓东..基于棉花U6启动子的海岛棉CRISPR/Cas9基因组编辑体系的建立[J].作物学报,2018,44(2):227-235,9.基金项目
本研究由国家自然科学基金项目(31660433)和新疆维吾尔自治区研究生科研创新项目(XJGRI2016055)资助.This study was supported by the National Natural Science Foundation of China(31660433)and the Innovation Research Program for Gradu-ate Students in Xinjiang(XJGRI2016055). （31660433）
