Cloning and Functional Analysis of Tobacco Polyphenol Oxidase NtPPO1-2 Gene

doi:10.13560/j.cnki.biotech.bull.1985.2025-0456

Abstract

Abstract:

Objective Polyphenol oxidase (PPO), a copper-binding enzyme, plays a significant role in plant growth, development, and defense against biotic and abiotic stresses. This study is aimed to clone and functionally analyze the NtPPO1-2 gene from cultivated tobacco (Nicotiana tabacum L.) K326 to lay the foundation for understanding polyphenol metabolism regulatory mechanisms in tobacco. Method Using cultivated tobacco K326 as the material, a novel PPO gene NtPPO1-2 was obtained through homologous cloning. Bioinformatics and gene expression analyses were performed. An overexpression vector was constructed, and transgenic plants were generated via Agrobacterium-mediated transformation. Phenotypic observations of the T₁ generation, PPO enzyme activity assays, and quantification of chlorogenic acid and rutin contents were conducted. Result The full-length of NtPPO1-2 is 1 821 bp, encoding 606 amino acids. Amino acid sequence alignment and domain analysis revealed that NtPPO1-2 shares the highest amino acid similarity with NsylPPO from Nicotiana sylvestris and contains conserved tyrosinase, PPO1_DWL, and PPO1_KFDV domains. The phenotype of the NtPPO1-2 T₁ overexpressing plants shows a reduced number of flower buds compared to the cultivated tobacco variety K326. Additionally, PPO enzyme activity was significantly elevated in overexpression plants, while the contents of major phenolic compounds, chlorogenic acid and rutin, were markedly reduced. Conclusion The NtPPO1-2 gene is successfully cloned from cultivated tobacco K326. Its overexpression significantly enhances PPO activity, reduces chlorogenic acid and rutin levels, and alteres plant phenotypes.

Key words: tobacco, polyphenol oxidase, NtPPO1-2 gene, gene cloning, polyphenol content

LIU Meng-ru, XIA Lin, LIU Rui-xia, YAN Xin-ke, YANG Jun, ZHANG Jing-hua, WU Ming-zhu. Cloning and Functional Analysis of Tobacco Polyphenol Oxidase NtPPO1-2 Gene[J]. Biotechnology Bulletin, 2026, 42(2): 218-227.

Figures/Tables 10

Table 1 Primer sequences used in this study

引物名称 Primer name	碱基序列 Base sequence （5′‒3′）	用途 Usage
NtPPO1-2-F	CGCCATCTCTTCTTCATCT	基因克隆
NtPPO1-2-R	CAATCAATCCTCAAGCACAA	Cloning of gene
NtPPO1-2-Q-F	ACCATAGTTTCCAAGTCTCA	NtPPO1-2基因RT-qPCR检测
NtPPO1-2-Q-R	ATGCCTCCTAATCCAAGTAG	RT-qPCR detecton of NtPPO1-2 gene
L25-Q-F	CCCCTCACCACAGAGTCTGC	RT-qPCR检测用内参引物
L25-Q-R	AAGGGTGTTGTTGTCCTCAATCTT	Internal reference primers for RT-qPCR analysis

Fig. 1 Electrophoresis pattern of PCR products of NtPPO1-2 geneM: Marker 2000. 1: Amplified product of NtPPO1-2

Fig. 2 Amino acid sequence alignment of PPO from N. tabacum, N. sylvestris, N. attenuata, N. tomentosiformis, Lycium barbarum, Solanum tuberosum, and Capsicum annuum

Fig. 3 Phylogenetic tree of plant PPO homologous protein

Fig. 4 Prediction of the secondary (A, B) and tertiary structures (C) of NtPPO1-2 protein

Fig. 5 Expression patterns of NtPPO1-2 gene in varioustobacco tissues at full-bloom stage

Fig. 6 Double digestion analysis of the NtPPO1-2 geneM: Marker 15000. 1: Double enzyme digestion of the sup1300 empty vector with Hind III and Nco I. 2: Hind III and Nco I double digestion of the sup1300-NtPPO1-2 plasmidND: Not detected. Lowercase letters above bars indicate significant differences in relative gene expression levels across tissues (P<0.05). The same below

Fig. 7 Analysis of gene expression of NtPPO1-2 in T1 transgenic plants

Fig. 8 Phenotypic changes (A) and enzyme activity analysis (B) of NtPPO1-2 gene overexpressing plants

Fig. 9 Analysis of chlorogenic acid and rutin content of plants with NtPPO1-2 overexpressed

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