doi:

DOI: 10.3724/SP.J.1006.2018.00227

Acta Agronomica Sinica (作物学报) 2018/44:2 PP.227-235

Establishment of CRISPR/Cas9 Genome Editing System Based on GbU6 Promoters in Cotton (Gossypium barbadense L.)


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 research.

Key words:cotton,protoplast,CRISPR/Cas9,genome editing

ReleaseDate:2018-03-08 10:13:39



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