烟草倍半萜合成酶基因NtEAS的体外功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2025-1234

摘要/Abstract

摘要：

目的萜类化合物在植物生长发育及逆境防御中发挥重要作用。旨在解析烟草倍半萜合成酶基因NtEAS（Nicotiana tabacum5-epi-aristolochene synthase）的生物学功能，为揭示萜类物质介导的抗病防御机制、推动烟草抗病育种提供理论依据。方法利用生物信息学方法分析NtEAS蛋白的特性、保守结构域、顺式作用元件及系统发育关系；利用RT-qPCR检测NtEAS在不同组织及病原菌胁迫下的表达模式；通过亚细胞定位技术明确NtEAS蛋白在烟草细胞中的亚细胞分布；通过原核表达系统体外表达并纯化NtEAS蛋白，鉴定NtEAS催化产物；采用菌丝生长速率法和滤纸片扩散法评估NtEAS催化产物对烟草青枯病菌和黑胫病菌的体外抑制活性，分析NtEAS在烟草黑胫病和青枯病抗性防御中的生物学功能。结果烟草倍半萜合成酶基因NtEAS开放阅读框全长1 647 bp，编码548个氨基酸，其编码蛋白定位于细胞核与细胞质，含有典型的萜类合成酶保守结构域“DDXXD”和“NST/DTE”。NtEAS主要在烟草根中高表达，且强烈响应烟草青枯病菌和黑胫病菌诱导。NtEAS能够以法尼基焦磷酸为底物催化生成倍半萜类化合物马兜铃烯，其催化产物对青枯病菌和黑胫病菌均具有显著的抑制活性，且这种抑制作用与其浓度呈正相关。结论 NtEAS基因及其催化产物可能同时参与烟草对青枯病和黑胫病的化学免疫防御过程。

关键词: 烟草, 倍半萜合成酶, NtEAS, 马兜铃烯, 青枯病, 黑胫病, 化学免疫防御

Abstract:

Objective Terpenoids play important roles in plant growth, development, and stress defense. This study aims to investigate the biological function of the tobacco sesquiterpene synthase gene NtEAS (Nicotiana tabacum5-epi-aristolochene synthase), and to provide a theoretical basis for revealing the terpene-mediated mechanisms of resistance and defense to disease and promoting molecular breeding in tobacco. Method Bioinformatics methods were employed to analyze the characteristics, conserved domains, cis-acting elements, and phylogenetic relationships of the NtEAS protein. RT-qPCR was used to detect the expression patterns of NtEAS in different tissues and under pathogen stress. Subcellular localization technology was applied to determine the distribution of NtEAS protein within cells. The NtEAS protein was expressed in vitro and purified using a prokaryotic expression system, and its enzymatic catalytic products were identified by GC-MS. The mycelial growth rate method and filter paper diffusion method were employed to evaluate the in vitro inhibitory activity of its catalytic products against tobacco bacterial wilt and black shank pathogens, in vitro prokaryotic expression and bacterial inhibitory activity analysis. Result The NtEAS has an open reading frame with a total length of 1647 bp, encoding 548 amino acids. Its encoded protein was located in the nucleus and cytoplasm and contains typical terpene synthase conserved domains “DDXXD” and “NST/DTE”. NtEAS was mainly highly expressed in tobacco roots and strongly induced by Ralstonia solanacearum and Phytophthora nicotianae. NtEAS can catalyze the formation of sesquiterpenoid aristolochene with farnesyl pyrophosphate as a substrate. The catalytic product exhibits significant inhibitory activity against both R. solanacearum and P. nicotianae, and this inhibitory effect is positively correlated with its concentrations. Conclusion NtEAS gene and its catalytic product may be involved in the chemical immune defense process of tobacco against bacterial wilt and black shank.

Key words: Nicotiana tabacum, sesquiterpene synthase, NtEAS, aristolochene, bacterial wilt, black shank, chemical immune defense

郑晓雨, 汪忆雪, 赵晓敏, 吴君章, 任洁馨, 钱亚新, 肖志亮, 刘正文, 潘晓薇, 李依婷. 烟草倍半萜合成酶基因NtEAS的体外功能鉴定[J]. 生物技术通报, 2026, 42(8): 197-206.

ZHENG Xiao-yu, WANG Yi-xue, ZHAO Xiao-min, WU Jun-zhang, REN Jie-xin, QIAN Ya-xin, XIAO Zhi-liang, LIU Zheng-wen, PAN Xiao-wei, LI Yi-ting. In vitro Functional Identification of Tobacco Sesquiterpene Synthase Gene NtEAS[J]. Biotechnology Bulletin, 2026, 42(8): 197-206.

图/表 13

表1 NtEAS的RT-qPCR引物

Table 1 RT-qPCR primers for NtEAS

引物 Primer	引物序列 Primer sequence （5′‒3′）
NtEAS-qF	TCTGCCACGGAGCAAGATTT
NtEAS-qR	TCTGTAGAGACGGGAGTGGG
Actin-qF	CAAGGAAATCACCGCTTTGG
Actin-qR	AAGGGATGCGAGGATGGA

图1 NtEAS蛋白的保守结构域分析方框标注的为萜烯合酶的保守结构域“DDXXD”和“NST/DTE”。CaEAS：甜椒马兜铃烯合酶；SiTPS9：番茄β-榄香烯合成酶；AtTPS21：拟南芥石竹烯合酶；ZmTPS1：玉米（E）-β-法尼烯合成酶

Fig. 1 Conserved domain analysis of NtEAS proteinsConserved domains of terpene synthase “DDXXD” and “NST/DTE” are marked in the box. CaEAS: Aristolochene synthase of bell pepper (Capsicum annuum L.); SiTPS9: Tomato (Solanum lycopersicum L.) β-elemene synthase; AtTPS21: Arabidopsis caryophyllene synthase; ZmTPS1: corn (E)-β-farnesene synthase

图2 NtEAS与拟南芥萜类合成酶家族的系统进化分析

Fig. 2 Phylogenetic analysis of NtEAS and Arabidopsis terpene synthase family

表2 NtEAS基因启动子中抗病相关顺式作用元件分析

Table 2 Analysis of NtEAS gene promoter cis-acting elements related to disease resistance

元件 Element	序列 Sequence （5'‒3'）	功能（响应信号/通路） Function （response signal/pathway）	可能结合的转录因子 Possible binding transcription factors	位置 Position
W-box	（T）TGAC（C/T）	响应病原体侵染，参与水杨酸（SA）或茉莉酸（JA）信号通路	WRKY转录因子家族	-363 ‒ -367、-605 ‒ -609、-739 ‒ -743、-828 ‒ -832
GCC-box	GCCGCC	响应乙烯（ET）信号，参与防御基因激活	ERF/AP2转录因子家族	-1 784 ‒ -1 789
JERE	CGTCA/TGACG	响应茉莉酸（JA）信号，激活防御相关基因表达	MYC、JAM等JA信号通路转录因子	-311 ‒ -315、-740 ‒ -744
P-box	CCTTTTG	参与苯丙烷类代谢途径（如植保素合成）	MYB转录因子	-855 ‒ -861

图3 NtEAS组织表达模式不同小写字母表示处理间在P<0.05水平上差异显著。下同

Fig. 3 Tissue expression pattern of NtEASDifferent lowercase letters indicate significant differences at the level of P<0.05. The same below

图4 NtEAS的亚细胞定位

Fig. 4 Subcellular location of NtEAS

图5 NtEAS的原核表达M：Marker；1‒2：NtEAS粗酶的上清（1）及沉淀（2）；3：纯化后的NtEAS蛋白。红色箭头指示为NtEAS蛋白

Fig. 5 Prokaryotic expression of NtEASM: Marker; 1‒2: supernatant (1) and precipitation (2) of NtEAS crude enzyme; 3: purified NtEAS protein. The red arrow indicates the NtEAS protein

图6 NtEAS的酶学功能鉴定A：纯化的NtEAS与FPP底物体外酶催化反应的GC-MS图；B和C：无底物（B）和无酶（C）反应体系的GC-MS图

Fig. 6 Enzymatic function identification of NtEASA: GC-MS diagram of the catalytic reaction between purified NtEAS and FPP substrate enzymes; B and C: GC-MS diagrams of substrate-free (B) and enzyme-free (C) reaction systems

图7 纯化的NtEAS催化FPP反应产物的GC-MS分子碎片质谱图

Fig. 7 GC-MS molecular fragment mass spectra of purified NtEAS-catalyzed FPP reaction products

图8 NtEAS响应青枯病菌胁迫的表达分析A：接种（RS）和不接种（CK）青枯病菌的感病种质C035中，NtEAS基因的FPKM值；B：接种（RS）和不接种（CK）青枯病菌的抗病种质C244中，NtEAS基因的FPKM值；C：在感病种质C035和抗病种质C244中，NtEAS基因FPKM增幅对比；D：接种青枯病菌后，感病种质红花大金元与抗病种质琼中五指山一号根中NtEAS的相对表达量

Fig. 8 Expression analysis of NtEAS in response to bacterial wilt stressA: FPKM values of NtEAS gene in C035 in the susceptible germplasm of inoculated (RS) and non-inoculated (CK) granville wilt; B: FPKM value of NtEAS gene in disease resistant germplasm C244 of inoculated (RS) and non-inoculated (CK) granville wilt; C: comparison of FPKM amplification of NtEAS gene in susceptible germplasm C035 and disease-resistant germplasm C244; D: the relative expression of NtEAS in the roots of the susceptible germplasm safflower Dajinyuan and the disease-resistant germplasm Qiongzhong Wuzhishan No. 1 after inoculation granville wilt

图9 NtEAS催化产物对青枯病菌的抑菌效果分析

Fig. 9 Analysis of the antibacterial effect of NtEAS catalytic products on bacterial wilt

图10 NtEAS响应黑胫病菌胁迫的表达模式A：感病种质小黄金1025（XHJ）与抗病种质Beinhart 1000-1（BH）接种黑胫病菌后，NtEAS在转录组中的FPKM值；B：接种黑胫病菌后，中抗种质K326根中NtEAS的相对表达量

Fig. 10 Expression pattern of NtEAS in response to black shank pathogen stressA: FPKM values of NtEAS in the transcriptome after inoculation with disease-resistant germplasmLittle Gold 1025 (XHJ) and disease-resistant germplasm Beinhart 1000-1 (BH). B: Relative expression of NtEAS in the roots of medium-resistant germplasm K326 after inoculation with black shank

图11 NtEAS催化产物对黑胫病菌的抑菌效果分析

Fig. 11 Analysis of the antibacterial effect of NtEAS catalytic products on black shank pathogen

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