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 Promoters in Cotton(Gossypium barbadense L.)

CRISPR/Cas9基因组编辑技术是基因功能研究的一种强有力的工具,目前已在许多生物体中成功实现内源靶向基因的突变。利用已克隆的海岛棉新海16的2个U6启动子,分别构建带有新海16内源基因(GbGGB和GbERA1)靶位点DNA片段的CRISPR/Cas9基因编辑载体。以新海16的胚性愈伤组织为供试材料,制备海岛棉的原生质体。通过PCR方法大量富集构建好的CRISPR/Cas9基因编辑载体的核心片段(包括GbU6::sg RNA和CAMV35S::Cas9两部分),并利用PEG法转化海岛棉的原生质体。对原生质体基因组DNA进行酶切后PCR,成功检测到内源靶基因的突变现象。对PCR产物进行克隆测序,结果显示序列突变的类型主要以碱基替换为主,少数为碱基缺失。结果表明基于海岛棉U6启动子的CRISPR/Cas9基因编辑系统能在海岛棉中实现靶向基因编辑的功能,为棉花功能基因组学研究提供了重要的技术基础。

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 research.