油楠SgTPS7的克隆及其在萜类生物合成和非生物胁迫中的功能

doi:10.13560/j.cnki.biotech.bull.1985.2024-0237

摘要/Abstract

摘要：

【目的】萜类化合物在植物生理和防御中发挥着重要作用。萜类合成酶基因（TPS）是萜类生物合成途径中的关键酶，热带泌油树种油楠（Sindora glabra）茎部含有大量萜类树脂油，探究油楠SgTPS在萜类物质合成和非生物胁迫响应中的功能，为通过合成生物学生产萜类提供重要基因资源，有助于拓展植物萜类合成途径与调控机制的认识。【方法】利用油楠基因组和转录组数据从油楠茎部组织克隆SgTPS7，并对其进行生物信息学分析；表达纯化SgTPS7蛋白利用GC-MS鉴定其催化功能；采用RT-qPCR技术分析SgTPS7的表达模式。【结果】油楠SgTPS7编码区序列全长1 650 bp。经同源比对分析发现，SgTPS7与苏木亚科古巴香胶树（Copaifera officinalis）CoTPS2的相似性最高，达92.3%，与其他科属植物的亲缘关系较远，属于苏木亚科特异的TPS基因。体外酶活试验表明，SgTPS7主要催化法尼基焦磷酸产生大根香叶烯D，同时催化香叶基焦磷酸产生芳樟醇。在非生物胁迫处理条件下，SgTPS7的表达差异显著。高温胁迫处理后，SgTPS7在叶片和茎中的表达均在24 h达到峰值，而在根部的表达则呈下降趋势；长期干旱处理显著影响SgTPS7的表达，在处理5 d后SgTPS7在叶和茎中的表达均达到最高，而根部SgTPS7的表达在10 d后达到最高。【结论】SgTPS7为多功能萜类合成酶基因，其在响应高温和干旱两种非生物胁迫中可能具有重要的作用。

关键词: 萜类合成酶, 酶活, 非生物胁迫, 油楠

Abstract:

【Objective】Terpenes play important roles in plant physiology and defense. The terpene synthase gene（TPS）is a key enzyme in the terpene biosynthetic pathway. The stems of the tropical oil-secreting tree species Sindora glabra contain a large amount of terpenoid resin oil. This study explores the role of S. glabra SgTPS in terpene synthesis and abiotic stress response. These will provide important genetic resources for the production of terpenes through synthetic biology, and help expand the understanding of plant terpene biosynthesis pathways and regulatory mechanisms.【Method】The SgTPS7 gene was cloned from the stem tissue of S. glabra using genome and transcriptome data, and bioinformatics analysis was performed on it. The SgTPS7 protein was expressed and purified and its catalytic function was identified using GC-MS, and RT-qPCR technology was used to analyze the expression pattern of SgTPS7.【Result】The full length sequence of the coding region of SgTPS7 of S. glabra was 1 650 bp. Homology comparison analysis found that SgTPS7 had the highest similarity with CoTPS2 of Copaifera officinalis, at 92.3%. It was distantly related to plants of other families and genera, and it belonged to the unique TPS genes of Caesalpinioideae. In vitro enzyme activity experiments showed that SgTPS7 mainly catalyzed farnesyl pyrophosphate to produce germacrene D, and catalyzed geranyl pyrophosphate to produce linalool. The expression of SgTPS7 gene was significantly different under different stress treatment conditions. After treatment with high-temperature stress, the expression of SgTPS7 in the leaves and stems reached the peak at 24 h, while the expression in the roots showed a downward trend. Long-term drought treatment significantly affected the expression of SgTPS7, and the expression of SgTPS7 in the leaves and stems reached the highest after 5 days of treatment, while the expression of SgTPS7 in the roots reached the highest after 10 d.【Conclusion】SgTPS7 is a multifunctional terpene synthase gene, which plays an important role in responding to abiotic stresses such as high temperature and drought.

Key words: terpene synthase, enzymatic activity, abiotic stress, Sindora glabra

余纽, 柳帆, 杨锦昌. 油楠SgTPS7的克隆及其在萜类生物合成和非生物胁迫中的功能[J]. 生物技术通报, 2024, 40(8): 164-173.

YU Niu, LIU Fan, YANG Jin-chang. Cloning of SgTPS7 in Sindora glabra and Its Function in Terpene Synthesis and Abiotic Stress[J]. Biotechnology Bulletin, 2024, 40(8): 164-173.

图/表 7

表1 引物序列

Table 1 Primers’ sequences

名称 Primer name	序列 Primer sequence（5'-3'）
SgTPS7-F	ATGACTAGATCCACAGCAGGC
SgTPS7-R	CTATTTATTGTGATCAATTGCTATGG
qSgTPS7-F	GCATATCTCGGTATCCGTGG
qSgTPS7-R	GCAGATCATTCCGAGAATCC
Action-2-F	CATGAAGTGTGATGTGGATA
Action-2-R	CCTTGCTCATTCTATCAGCA

图1 SgTPS7 的PCR产物琼脂糖凝胶电泳

Fig. 1 Agarose gel electrophoresis of SgTPS7 PCR products

图2 油楠SgTPSs氨基酸序列对比及结构域分析

Fig. 2 Comparison of amino acid sequences and domain analysis of SgTPSs from S. glabra

图3 不同植物TPS同源蛋白序列的系统进化树分析 PtTPS：毛果杨；CoTPS：古巴香胶树；AtTPS：拟南芥；StTPS：马铃薯

Fig. 3 Phylogenetic tree analysis of TPS homologous protein sequences in different plants PtTPS: Populus trichocarpa; CoTPS: Copaifera officinalis; AtTPS: Arabidopsis thaliana; StTPS: Solanum Tuberosum

图4 SgTPS7蛋白表达纯化分析 A：SgTPS7蛋白表达[M：protein marker；0：对照（不加IPTG）：1：15℃诱导16 h；2：37℃诱导16 h]；B：SgTPS7蛋白可溶性（1：上清；2：沉淀）；C：SgTPS7蛋白上清纯化结果（1：上清同Ni-IDA 孵育后流出液；2-4：100 mmol/L咪唑洗脱组分；5-6：300 mmol/L咪唑洗脱组分）；D：SgTPS7纯化蛋白[1：BSA（2.00 μg）；2：SgTPS7蛋白（2.00 μg）]

Fig. 4 Expression and purification analysis of SgTPS7 protein A: SgTPS7 protein expression[M: protein marker; 0: control（without IPTG）; 1: 15℃ for 16 h; 2: 37℃ for 16 h]. B: SgTPS7 protein solubility（1: supernatant; 2: precipitation）. C: SgTPS7 protein purification（1: outflow; 2-4: 100 mmol/L imidazole elution; 5-6: 300 mmol/L imidazole elution）. D: Purified SgTPS7 protein[1: BSA（2.00 μg）; 2: SgTPS7 protein（2.00 μg）]

图5 GC-MS分析SgTPS7的催化产物 1、2、3、4、5分别为β-衣兰烯、β-可巴烯、大根香叶烯D、橙花叔醇和芳樟醇

Fig. 5 GC-MS analysis of enzymatic products of SgTPS7 protein 1, 2, 3, 4 and 5 are ylangene, β-copaene, germacrene D, nerolidol, and linalool, respectively

图6 不同胁迫处理下SgTPS7的表达水平不同小写字母表示在同一组织中不同时间点SgTPS7表达量的统计学差异显著（P<0.05）

Fig. 6 Expressions of SgTPS7 under various stresses Different lowercase letters indicate significant statistical differences of SgTPS7 expressions from the same tissue at different time points（P<0.05）

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