烟草多酚氧化酶NtPPO1-2基因克隆及功能分析

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

摘要/Abstract

摘要：

目的多酚氧化酶（polyphenol oxidase, PPO）是一种铜离子结合酶，其在植物的生长发育、抵御生物和非生物胁迫中具有重要作用。克隆栽培烟草（Nicotiana tabacum L.）K326 NtPPO1-2基因并分析其功能，为烟草多酚代谢调控机制研究奠定基础。方法以栽培烟草K326为材料，通过同源克隆获得1个新的多酚氧化酶（PPO）基因，命名为NtPPO1-2。进行生物信息学分析和基因表达分析；构建过表达载体并通过农杆菌介导法获得转基因植株；观察T₁代表型、测定PPO酶活性及绿原酸和芦丁含量。结果 NtPPO1-2基因全长1 821 bp，编码606个氨基酸。氨基酸序列比对和结构域分析结果显示，烟草NtPPO1-2与林烟草NsylPPO氨基酸序列相似度最高，并且NtPPO1-2包含保守的酪氨酸酶（tyrosinase）、PPO1_DWL及PPO1_KFDV结构域。T₁代过表达植株的表型与栽培烟草K326相比，花蕾数目减少。且NtPPO1-2基因过表达植株多酚氧化酶的活性显著升高，烟草主要的多酚物质绿原酸和芦丁的含量显著下降。结论从栽培烟草K326中成功克隆到NtPPO1-2基因，其过表达通过增强PPO活性显著降低绿原酸和芦丁含量，并改变烟草植株表型。

关键词: 烟草, 多酚氧化酶, NtPPO1-2基因, 基因克隆, 多酚含量

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

刘梦茹, 夏琳, 刘瑞霞, 闫新可, 杨军, 张菁华, 武明珠. 烟草多酚氧化酶NtPPO1-2基因克隆及功能分析[J]. 生物技术通报, 2026, 42(2): 218-227.

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.

图/表 10

表1 本研究所用引物序列

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

图1 NtPPO1-2基因PCR产物电泳图M：Marker 2000；1：NtPPO1-2基因扩增产物

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

图2 烟草与林烟草、野生烟草、绒毛状烟草、枸杞、马铃薯以及辣椒PPO的氨基酸序列比对

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

图3 植物PPO同源蛋白系统进化树

Fig. 3 Phylogenetic tree of plant PPO homologous protein

图4 NtPPO1-2蛋白二级（A、B）和三级结构（C）预测

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

图5 盛花期烟草不同组织中NtPPO1-2基因的表达模式

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

图6 NtPPO1-2基因双酶切验证M：Marker 15000；1：Hind III和Nco I双酶切sup1300空载体；2：Hind III和Nco I双酶切sup1300-NtPPO1-2质粒ND：未检测到；小写字母表示不同组织间基因相对表达量差异显著（P<0.05），下同

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

图7 T1代转基因植株NtPPO1-2基因表达分析

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

图8 NtPPO1-2基因过表达植株表型变化（A）和酶活分析（B）

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

图9 NtPPO1-2基因过表达植株的绿原酸和芦丁含量分析

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

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