CRISPR/Cas9-mediated Targeted Knockout of Polyphenol Oxidase NtPPO1 Gene in Nicotiana tabacum

doi:10.13560/j.cnki.biotech.bull.1985.2018-0560

Abstract

Abstract: Polyphenol oxidases（PPOs）have many important roles in plants. In order to studying PPOs functions in Nicotiana tabacum,a PPO（Sequence ID：XM_016608009.1）was selected from the GenBank and named as NtPPO1. Then,the CRISPR/Cas9-mediated genome-editing tool was utilized to knockout the tobacco NtPPO1after NtPPO1 cDNA sequence amplified by PCR and determined by clone,and NtPPO1 relative transcript levels were analyzed by qPCR. As result,the full length of NtPPO1 cDNA was 1 746bp,and there were two kinds of alternative splicing in NtPPO1 transcripts. Additionally,1 bp or 2 bp deletion and 1 bp insertion at the target site in the NtPPO1 sequence of T₂homozygous mutants occurred. Compared to the wild control plants,NtPPO1 relative expression in T₂mutants by qPCR significantly reduced. Meanwhile,there were no obvious difference on the appearance between the T₂mutants and wild ones of N. tabacum.

Key words: Nicotiana tabacum, CRISPR/Cas9, NtPPO1, genome editing, polyphenol oxidase（PPO）

YAO Heng, YANG Da-hai, BAI Ge, XIE He. CRISPR/Cas9-mediated Targeted Knockout of Polyphenol Oxidase NtPPO1 Gene in Nicotiana tabacum[J]. Biotechnology Bulletin, 2018, 34(11): 97-102.

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