Cloning of SgTPS7 in Sindora glabra and Its Function in Terpene Synthesis and Abiotic Stress

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

Abstract

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

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.

Figures/Tables 7

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

Fig. 1 Agarose gel electrophoresis of SgTPS7 PCR products

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

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

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）]

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

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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