利用CRISPR/Cas9技术定点敲除烟草多酚氧化酶基因NtPPO1

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

摘要/Abstract

摘要： 植物多酚氧化酶具有多种重要的生理功能。为研究烟草中多酚氧化酶基因功能,从GenBank中挑选一个烟草多酚氧化酶基因（基因登录号为XM_016608009.1）,命名为NtPPO1。NtPPO1编码序列经PCR扩增、克隆测序验证后,采用CRISPR/Cas9技术定点敲除NtPPO1,并利用实时荧光定量PCR检验基因敲除效果。研究表明烟草NtPPO1全长1 746 bp,存在2种可变剪切方式。经测序与分析发现,T₂突变体中的NtPPO1序列在靶位点处发生了1个、2个碱基的缺失突变与1个碱基的插入突变。实时荧光定量PCR检测结果显示,与对照相比,T₂突变体中NtPPO1表达水平显著下降。烟株外观显示NtPPO1突变体与野生型对照之间无明显差异。

关键词: 烟草, CRISPR/Cas9, NtPPO1, 基因组编辑, 多酚氧化酶

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）

姚恒, 杨大海, 白戈, 谢贺. 利用CRISPR/Cas9技术定点敲除烟草多酚氧化酶基因NtPPO1[J]. 生物技术通报, 2018, 34(11): 97-102.

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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