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植物学报 >

2025 , Vol. 60 >Issue 3: 363 - 376

DOI: https://doi.org/10.11983/CBB24139

研究论文

甘蓝中催化NMN降解生成NR的5′-核苷酸酶基因克隆和功能分析

  • 刘茹 ,
  • 李阳 ,
  • 唐兆成 ,
  • 郝婷婷 ,
  • 张保龙
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  • 1南京农业大学生命科学学院, 南京 210014
    2江苏省农业科学院种质资源与生物技术研究所, 南京 210014
*张保龙, 博士, 研究员。中国农业生物技术学会常务理事, 江苏省遗传学会理事。科技部中青年科技创新领军人才, 江苏省333人才第二层次, 江苏省六大高峰人才。长期从事酶蛋白结构解析、酶的定向进化以及酶的催化反应机制等研究。以通讯作者身份在Nature Communications、Plant Cell、Plant Biotechnology Journal、PLoS Pathogens、Journal of Agricultural and Food Chemistry等权威期刊上发表论文20余篇。荣获国家科学技术进步二等奖1项, 江苏省科学技术进步二等奖2项, 中华农业科技奖1项。E-mail: zhbl2248@hotmail.com;
郝婷婷, 博士, 助理研究员。主要研究方向为天然产物代谢途径。以第一作者或通讯作者身份在Nature Communications、Food Research International等期刊发表论文10余篇。主持省级项目1项。E-mail: 17812067912@126.com

收稿日期: 2024-09-09

  录用日期: 2024-12-14

  网络出版日期: 2024-12-17

基金资助

生物育种钟山实验室项目(BM2022008-02)

收起

Cloning and Functional Analysis of the 5'-nucleotidase Genes Catalyze NMN Degradation to NR in Brassica oleracea var. acephala

  • Liu Ru ,
  • Li Yang ,
  • Tang Zhaocheng ,
  • Hao Tingting ,
  • Zhang Baolong
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  • 1College of Life Sciences, Nanjing Agricultural University, Nanjing 210014, China
    2Institute of Germplasm Resources and Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

Received date: 2024-09-09

  Accepted date: 2024-12-14

  Online published: 2024-12-17

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摘要

烟酰胺单核苷酸(NMN)具有抗癌和抗衰老等重要生物活性。植物中NMN含量低, 阻断其降解途径是提高NMN含量的有效方式, 而植物中NMN降解途径尚不清晰。从羽衣甘蓝(Brassica oleracea var. acephala)中克隆到8个5′-核苷酸酶候选基因, 对其编码产物进行生物信息学分析, 利用大肠杆菌表达系统纯化蛋白。系统发育分析表明, 5′-核苷酸酶在植物中保守存在, 暗示其在调控核苷酸代谢中可能发挥重要作用。通过体外酶促反应分析了这些5′-核苷酸酶的催化性能, 结果表明, 羽衣甘蓝中5′-核苷酸酶BolN2、BolN5-X1和BolN6能够催化NMN降解生成烟酰胺核糖(NR)。此外, BolN2、BolN5和BolN6还能够催化烟酸单核苷酸、嘌呤和嘧啶核苷酸水解, 表明其具有广泛的底物适应性。研究揭示了羽衣甘蓝5′-核苷酸酶的催化特性, 为阐明甘蓝中NMN的降解途径, 进而创制高NMN含量的甘蓝新种质奠定了基础。

关键词： 羽衣甘蓝; 烟酰胺单核苷酸; 5′-核苷酸酶; 功能验证

本文引用格式

刘茹 , 李阳 , 唐兆成 , 郝婷婷 , 张保龙 . 甘蓝中催化NMN降解生成NR的5′-核苷酸酶基因克隆和功能分析[J]. 植物学报, 2025 , 60(3) : 363 -376 . DOI: 10.11983/CBB24139

Abstract

INTRODUCTION: Nicotinamide mononucleotide (NMN) has important biological activities such as anti-cancer, anti-aging and improving crop stress resistance, and its importance as a nutritional health product has been established. However, the content of NMN in plants is low, and the metabolic pathway of NMN degradation is poorly understood. It has been reported that 5'-nucleotidases can catalyze the dephosphorylation of NMN in Saccharomyces cerevisiae. At present, 5'-nucleotidases have been isolated in plants, but whether they can catalyze the degradation of NMN remains unclear. Edible kale (Brassica oleracea var. acephala) has high nutritional value. It is important to analyze the metabolic pathway of NMN in kale and increase the content of NMN by blocking the degradation pathway.

RATIONALE: The degradation of NMN in plants is closely related to the NAD+ remediation synthesis (pyridine nucleotide cycle) pathway. Compared with bacteria and mammals, studies on the biosynthetic pathway of NAD+ remediation in plants mainly use isotope tracer method, lack specific gene and function analysis, and only a few related studies have been reported in plants. Eight 5'-nucleotidase genes were cloned from B. oleraceavar. acephala, heterologous expression of them was performed by Escherichia coli expression system, and the catalytic properties of 5'-nucleotidase were investigated by enzymological means in vitro.

RESULTS: In this study, ten 5'-nucleotidase genes were retrieved from the genome of B. oleracea. Based on these sequences, eight 5'-nucleotidase candidate genes were successfully cloned from B. oleracea var. acephala, which laid a foundation for further revealing the degradation pathway of NMN. Phylogenetic analysis revealed that 5'-nucleotidase is conserved in plants, suggesting that it may play an important role in plant nucleotide metabolism. The catalytic properties of 5'-nucleotides in kale were investigated by using the expression system of E. coli. In vitro enzymatic experiments showed that 5'-nucleotides can catalyze purine, pyrimidine and pyridine nucleotides, and have a wide range of substrate adaptability. Specifically, BolN2, BolN5-X1 and BolN6 can catalyze the dephosphorylation of NMN to the generation of NR, which proves that 5'-nucleotidase can catalyze the degradation of NMN in plants. In addition, BolN2, BolN5 and BolN6 can catalyze the hydrolysis of pyridine nucleotides NaMN, purine and pyrimidine nucleotides (including AMP, GMP, CMP and UMP). However, BolN7 and BolN8 had only weak catalytic activity against GMP.

CONCLUSION: The 5'-nucleotidase gene from the HAD and SurE families of B. oleracea var. acephalawas cloned and phylogenetic analysis showed that it was conserved in plants. The results of enzymatic reaction in vitro showed that BolN2, BolN5-X1 and BolN6 could catalyze the degradation of NMN to produce NR. In addition, BolN2, BolN5 and BolN6 have catalytic effects on NaNM, purine and pyrimidine nucleotides. This study further enhanced our understanding of the NMN metabolic pathway in kale, and provided a theoretical basis for creating edible kale new germplasm with high NMN content.



Cloning and functional analysis of 5'-nucleotidase gene catalyzing NMN degradation to NR inBrassica oleraceavar. acephala.The 5'-nucleotidase gene of B. oleraceavar. acephala was successfully cloned and phylogenetic analysis showed that it was conserved in plants. By constructing prokaryotic expression vector, 5'-nucleotidase was expressed and purified in Escherichia coli. In vitro enzymatic experiments showed that 5'-nucleotidase could catalyze the dephosphorylation of NMN to NR, and had catalytic effects on NaMN, purine nucleotides (AMP, GMP) and pyrimidine nucleotides (CMP, UMP).

Key words： Brassica oleracea var. acephala; nicotinamide mononucleotide; 5′-nucleotidase; functional verification

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