编辑烤烟多酚氧化酶基因NtPPO8后的效应分析

  目的  探究NtPPO8基因在烟草中的功能以及对烟叶耐烤性和烤后烟质量的影响。  方法  以烤烟品种NC89为材料，利用CRISPR/Cas9技术对NtPPO8进行定向编辑，获得纯合突变植株，测定并分析原品种和突变体在叶龄60 d时的基因表达量和植物多酚氧化酶（Polyphenol oxidase，PPO）活性差异，探究NtPPO8基因对烘烤过程中的PPO活性以及烤后烟的多酚物质含量的影响。  结果  （1）共获得6株T₀代纯合突变体，突变均发生在靶位点上，为1个碱基的插入突变。（2）4个T₁代编辑株系的NtPPO8表达水平显著下降，所有编辑株系的PPO活性显著下降。（3）T₁代编辑株系烘烤过程中的PPO活性低于原品种，烤后中部叶总酚含量显著高于原品种。  结论  定向编辑NtPPO8基因后，降低了其在烟叶中的表达量，抑制了烟叶在烘烤过程中的PPO活性，有助于烤后烟多酚类物质的积累。 

Effect analysis of editing flue-cured tobacco polyphenol pxidase gene NtPPO8

  Objective  This study aims to explore the function of NtPPO8 gene in tobacco as well as its influence on tobacco leaf curing tolerance and quality of flue-cured tobacco.  Methods  Using flue-cured tobacco variety NC89 as material, NtPPO8 was directionally edited by CRISPR/Cas9 technology to obtain homozygous mutant plants. The differences in gene expression levels and plant polyphenol oxidase (PPO) activity between the original variety and the mutant at leaf age of 60 days were determined and analyzed, so as to explore the effect of NtPPO8 gene on the PPO activity during the curing process and the content of polyphenols in the tobacco after curing.  Results  (1) A total of 6 strains of T₀ homozygous mutants were obtained, all mutations occurred at the target site, which belong to 1 base insertion mutations. (2) The expression levels of NtPPO8 in the four T₁ generation edited lines were significantly decreased, and the PPO activities of all edited lines were significantly decreased. (3) The PPO activity of the edited lines in T₁ generation during curing was lower than that of the wild type, and the total phenolic content of the middle leaves after curing was significantly higher than that of the original variety.  Conclusion  Directional editing of the NtPPO8 gene reduced its expression in tobacco leaves, inhibited the PPO activity of tobacco leaves during the curing process, and contributed to the accumulation of polyphenols in tobacco after curing.