基于VIGS和CRISPR/Cas9技术的烟草NtPPO5基因功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1055

摘要/Abstract

摘要：

目的探究普通栽培烟草（Nicotiana tabacum L.）K326中多酚氧化酶（polyphenol oxidase, PPO）基因NtPPO5的序列特征、进化关系、组织表达模式，及其对PPO活性和总酚含量的调控作用。方法为了解析NtPPO5（基因登录号为Nta24g03870.1）的基因功能，本研究从普通栽培烟草品种K326中克隆了该基因。通过氨基酸序列比对和系统进化分析，明确了NtPPO5及其同源基因的进化关系。进一步检测了NtPPO5基因在烟草不同组织中的表达模式，并利用VIGS技术和CRISPR/Cas9系统分别对该基因进行沉默和敲除，获得了NtPPO5功能缺失型植株。在此基础上，分析了突变植株叶片中NtPPO5基因的表达水平、PPO活性以及总酚含量的变化。结果 NtPPO5基因所编码的氨基酸序列包含PPO家族典型的保守结构域，包括Tyrosinase、PPO1_DWL和PPO1_KFDV。系统进化分析显示，NtPPO5与绒毛状烟草NtomPPO及普通烟草NtPPO1处于同一进化分支，表明它们亲缘关系较近。组织表达分析表明，NtPPO5在根、茎、叶和花中均有表达，其中在叶片中的表达量最高。在VIGS沉默和CRISPR/Cas9敲除植株中，NtPPO5的表达水平和PPO活性均显著下降，而总酚含量则显著上升。结论 NtPPO5是PPO家族的典型成员，与NtomPPO和NtPPO1亲缘关系最近。该基因在叶片中呈现高表达，其表达水平与PPO活性呈正相关，而与总酚含量呈负相关。

关键词: 烟草, 多酚氧化酶, NtPPO5基因, VIGS, CRISPR/Cas9, 总酚含量

Abstract:

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.

Key words: tobacco, polyphenol oxidase, NtPPO5 gene, VIGS, CRISPR/Cas9, total phenolic content

彭朝凤, 夏琳, 刘瑞霞, 闫新可, 牧杨威, 刘梦茹, 杨军, 武明珠. 基于VIGS和CRISPR/Cas9技术的烟草NtPPO5基因功能分析[J]. 生物技术通报, 2026, 42(4): 251-262.

PENG Chao-feng, XIA Lin, LIU Rui-xia, YAN Xin-ke, MU Yang-wei, LIU Meng-ru, YANG Jun, WU Ming-zhu. Functional Analysis of the Tobacco NtPPO5 Gene Using VIGS and CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2026, 42(4): 251-262.

图/表 11

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	序列 Base sequence （5′‒3′）	用途 Usage
NtPPO-F	ATGGCTTCTTCATTTGTTC	基因克隆
NtPPO-R	TTAACAAGGGACCAACTG
NtPPO5-Q-F	TTCAAGCCACAACCAAGA	RT-qPCR检测NtPPO5基因
NtPPO5-Q-R	TCACATCCAATTCCACATTC	RT-qPCR检测NtPPO5基因
L25-F	CCCCTCACCACAGAGTCTGC	RT-qPCR检测内参基因
L25-R	AAGGGTGTTGTTGTCCTCAATCTT
NtPPO5-VIGS-F	TCGACGACAAGACCCTGCAGCCTCCTGTATCCAGATTTCG	构建VIGS载体
NtPPO5-VIGS-R	TGAGGAGAAGAGCCCTGCAGGATTCTCTCATAGAAGTATATGT
Cas-F	GGGATCCGAAGAAGTACGGC	基因编辑载体鉴定
Cas-R	TATTCTCAGCCTGCTCCCTG
NtPPO5-BJ-F	ggagtgagtacggtgtgcTTCCAAGTATCATGCAACCA	基因编辑植株突变位点鉴定
NtPPO5-BJ-R	gagttggatgctggatggGGATAGGAGGACAACAAGTG	基因编辑植株突变位点鉴定

图1 NtPPO5基因扩增产物电泳图M： DL2 000 DNA marker； I： NtPPO5基因PCR扩增产物

Fig. 1 Electrophoretogram of NtPPO5 gene amplified productM: DL2 000 DNA marker. 1: PCR amplification product of the NtPPO5 gene

图2 NtPPO5与其他茄科植物氨基酸序列比对

Fig. 2 Alignment of the amino acid sequences of NtPPO5 with other Solanaceae plants

图3 NtPPO5遗传进化分析

Fig. 3 Genetic evolutionary analysis of NtPPO5

图4 盛花期烟草不同组织NtPPO5基因表达分析不同小写字母表示组织间差异达到显著水平（P<0.05），下同。ND：未检出

Fig. 4 Analysis of NtPPO5 gene expression in different tissues during the tobacco flowering stageDifferent lowercase letters indicate significant differences among tissues (P<0.05). The same below. ND:None detected

图5 pTRV2-NtPPO5基因重组质粒检测A：pTRV2-NtPPO5菌液PCR检测结果（M：DL2 000 DNA marker；1-7：pTRV2-NtPPO5菌液）；B：pTRV2-NtPPO5质粒酶切检测结果（M：DL15 000 DNA marker；1：pTRV2-NtPPO5质粒）

Fig. 5 Detection of pTRV2-NtPPO5 gene recombinant plasmidA: PCR results of pTRV2-NtPPO5 bacterial liquid (M: DL2 000 DNA marker. Lane 1-7: pTRV2-NtPPO5 bacterial liquid). B: Restriction enzyme digestion results of pTRV2-NtPPO5 plasmid (M: DL15 000 DNA marker. Lane 1: pTRV2-NtPPO5 plasmid)

图6 NtPPO5基因沉默植株表型及基因相对表达分析A：空白对照（Con）与阳性对照（pTRV2-PDS）、阴性对照（pTRV2）植株及实验组（pTRV2-NtPPO5）植株侵染15 d后的表型；B：NtPPO5基因相对表达量分析

Fig. 6 Phenotypic characteristics and relative gene expressions of NtPPO5 gene silenced plantsA: Phenotype of blank control (Con), positive control (pTRV2-PDS), negative control (pTRV2), and experimental group (pTRV2-NtPPO5) plants at 15 d post-infiltration. B: Relative expression analysis of the NtPPO5 gene

图7 NtPPO5基因沉默植株PPO活性（A）及总酚含量分析（B）

Fig. 7 Analysis of polyphenol oxidase (PPO) activity (A) and total phenolic content (B) in NtPPO5 transgenic plants

图8 NtPPO5基因编辑植株测序结果分析

Fig. 8 Analysis of sequencing of NtPPO5 gene-edited plants

图9 CRISPR/Cas9-NtPPO5基因敲除载体检测A：脱靶基因序列示意图；B：脱靶基因测序结果比对

Fig. 9 Detection of CRISPR/Cas9-mediated knockout of the NtPPO5 gene vectorA: Schematic diagram of off-target gene sequences. B: Alignment of off-target gene sequencing results

图10 NtPPO5基因编辑植株表型（A）、烤烟表型（B）、PPO活性（C）、总酚（D）和绿原酸含量（E）分析

Fig. 10 Analysis of phenotype (A), cured tobacco phenotype (B), polyphenol oxidase activity (C), total phenolics (D), and chlorogenic acid content (E) in NtPPO5 gene-edited plants

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