Biotechnology Bulletin ›› 2018, Vol. 34 ›› Issue (11): 97-102.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0560

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

YAO Heng, YANG Da-hai, BAI Ge, XIE He   

  1. Yunnan Academy of Tobacco Agricultural Science,Kunming 650021
  • Received:2018-06-19 Online:2018-11-26 Published:2018-11-28

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 T2 homozygous mutants occurred. Compared to the wild control plants,NtPPO1 relative expression in T2 mutants by qPCR significantly reduced. Meanwhile,there were no obvious difference on the appearance between the T2 mutants and wild ones of N. tabacum.

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