Abstract:

Objective: The oleoresin in the stem of Sindora glabra contains rich sesquiterpenoids, while terpene synthase is one of the key enzymes in the terpene biosynthesis pathway. Cloning and expression analysis of S. glabra sesquiterpene synthase gene SgSTPS3 is helpful to explore the formation mechanism of S. glabra oleoresin, which will provide a new theoretical basis for analyzing the mechanisms of terpenoid biosynthesis and metabolism in forest trees. Method: Based on the previous transcriptome data, the sesquiterpene synthase gene SgSTPS3 was cloned from the stem of mature S. glabra, and analyzed by bioinformatics tools. Prokaryotic expression in Escherichia coli cells, and identification by-products by GC-MS. The expression levels of this gene at different ages (0.5, 1.5, 15, 30 years old) and in differet organs (root, phloem, xylem, tender leaves, old leaves) of S. glabra were also analyzed by quantitative real-time PCR. To analyze the response pattern of this gene to hormones, salicylic acid (SA), methyl jasmonate (MeJA), and the combination of SA+MeJA were applied. Result: The full-length cDNA sequence of SgSTPS3 gene including 1 701 bp was cloned and encoded 566 amino acids(GenBank accession number: MW345633). The multiple sequence alignments indicated that SgSTPS3 had the highest homology with Copaifera officenalis CoTPS3 and Copaifera langsdorffii ClTPS3, reaching 87.68%. Biochemical analysis showed that its protein was a multifunctional enzyme. When farnesyl pyrophosphate(FPP) was used as a substrate, it was mainly catalyzed and produced sesquiterpene cyclosativene, copaene and cis-β-copaene; when geranyl pyrophosphate(GPP) was used as the substrate, it was mainly produced monoterpene linalool. The expression level of SgSTPS3 gene was significantly different in different organs and at different ages: in different organs of same plant age, the highest expression level appeared in the root of 0.5 and 1.5 years old plants, and the hightest in the phloem of 15 and 30 years old plants; for the same organ at different ages, the expression of roots decreased with age, while the expression in the xylem increased with age; the expression in phloem of 30 years old plants was significantly higher than that of the other three ages, but there was no significant rule between young and old leaves. Hormone treatment was performed on the leaves of 2 years old S. glabra, the highest expression level of this gene appeared in 12 h after salicylic acid(SA, 50 μmol·L^-1) spraying, 35.07 folds compared to the control, and the lowest was in 72 h. In addition, its expression was highest at 6 h and lowest at 24 h after methyl jasmonate (MeJA, 100 μmol·L^-1) treatment. Moreover, the expression of SgSTPS3 gene was similar to that of the control group, under the treatment of SA(50 μmol·L^-1)+MeJA(100 μmol·L^-1), with a poor induction effect. Conclusion: SgSTPS3 was a sesquiterpene synthetase gene with multi-substrate enzyme catalytic function, and its expression level was higher in the root of seedlings and the phloem of adult plants. The regulation of different hormones on SgSTPS3 gene was significantly different, among which SA(50 μmol·L^-1) had the best induction effect, and MeJA (100 μmol·L^-1) came the second.

Key words: terpene synthetase gene, expression pattern, hormone regulation, Sindora glabra