In vitro Functional Identification of Tobacco Sesquiterpene Synthase Gene NtEAS

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

Abstract

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

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.

Figures/Tables 13

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

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

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

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

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

Fig. 4 Subcellular location of NtEAS

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

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

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

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

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

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

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

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