Functional Analysis of the Tobacco NtPPO5 Gene Using VIGS and CRISPR/Cas9 Technology

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

Abstract

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

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.

Figures/Tables 11

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

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

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

Fig. 3 Genetic evolutionary analysis of NtPPO5

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

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)

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

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

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

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

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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