基于VIGS和CRISPR/Cas9技术的烟草NtPPO5基因功能分析OA

[Objective]To investigate the sequence characteristics,phylogenetic relationships,tissue-specific expression patterns of the polyphenol oxidase gene NtPPO5 in flue-cured tobacco(Nicotiana tabacum L.)K326,and its regulatory effects on PPO activity and total phenol content.[Method]To elucidate the function of the NtPPO5 gene(Accession number is Nta24g03870.10),it was cloned from the common cultivated tobacco cultivar K326.Subsequently,phylogenetic and amino acid sequence analyses were conducted to clarify the evolutionary relationships between NtPPO5 and its homologs.Furthermore,the expression patterns of NtPPO5 in various tobacco tissues were examined.Using both VIGS and CRISPR/Cas9 systems,loss-of-function mutants of NtPPO5 were generated through gene silencing and knockout,respectively.Based on this,changes in NtPPO5 expression levels,PPO activity,and total phenolic content were analyzed in the leaves of the mutant plants.[Result]The amino acid sequence encoded by the NtPPO5 gene contained typical conserved domains of the PPO family,including tyrosinase,PPO1_DWL,and PPO1_KFDV.Phylogenetic analysis revealed that NtPPO5 clusters within the same clade as NtomPPO from Nicotiana tomentosiformis and NtPPO1 from N.tabacum,indicating a close evolutionary relationship.Tissue-specific expression analysis showed that NtPPO5 was expressed in the root,stem,leaf,and flower tissues,with the highest expression observed in leaves.In both VIGS-silenced and CRISPR/Cas9 knockout plants,the expression of NtPPO5 and PPO activity significantly decreased,while the total phenolic content markedly increased.[Conclusion]NtPPO5 is a typical member of the PPO family,closely related to NtomPPO and NtPPO1.This gene is highly expressed in the leaves,with its expression positively correlated with PPO activity and negatively correlated with total phenol content.