Construction of CRISPR/Cas9 Genome Editing Vectors Targeting Short Vegetative Phase in Rapeseed

doi:10.7668/hbnxb.2018.03.006

Abstract

Abstract: Targeted genome editing via CRISPR/Cas9 is emerging as a powerful tool for molecular modification in recent years,by which the target gene will lose functions because of deletion,insertion or substitution of several nucleotides in specific sites. It has been widely used to researches on gene function. To deeply elucidate the biological function of Short vegetative phase (SVP)in rapeseed(Brassica napus L.)by entire or partial knock-outs,totally twelve sgRNA seed sequences targeting four homologous copies of SVP distributed in Zhongshuang No.11 genome(Brassica napus reference genome,v4.1)were designed by using software CRISPR-P 2.0,and then six dual sgRNAs CRISPR/Cas9 genome editing constructs were developed employing pYLCRISPR-Cas9P35S-H as backbone and AtU3b or AtU3d as promoter with Golden Gate Cloning technology. Sequencing results demonstrated that all the recombinant plasmids showed the correct nucleotides and expression cassette assembly,which lay a solid foundation for further protoplast transient expression assay and Agrobacterium -mediated transformation of rapeseed. Meanwhile,this paper provides a strategy to study functions of multiple homologous genes in plants applying CRISPR/Cas9 genome editing system.

Key words: Brassica napus, Short vegetative phase gene, CRISPR/Cas9, Genome editing construct

CLC Number:

ZHAO Heng, ZHANG Hong, LIAO Fangli, LI Gang, ZHAO Fuyong. Construction of CRISPR/Cas9 Genome Editing Vectors Targeting Short Vegetative Phase in Rapeseed[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(3): 31-37. doi: 10.7668/hbnxb.2018.03.006.

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