Cloning, prokaryotic expression, and functional identification of a sesquiterpene synthase gene (AsSS4) from Aquilaria sinensis.
- Author:
Liang LIANG
;
Qing-Mei GUO
;
Zheng ZHANG
;
Yan-Hong XU
;
Xiao-Min HAN
;
Juan LIU
- Publication Type:Journal Article
- MeSH:
Alkyl and Aryl Transferases;
biosynthesis;
genetics;
Azulenes;
Cloning, Molecular;
DNA, Complementary;
Escherichia coli;
Open Reading Frames;
Polyisoprenyl Phosphates;
Recombinant Proteins;
biosynthesis;
Sesquiterpenes;
metabolism;
Sesquiterpenes, Guaiane;
Thymelaeaceae;
enzymology;
genetics
- From:
Acta Pharmaceutica Sinica
2014;49(12):1724-1729
- CountryChina
- Language:Chinese
-
Abstract:
A sesquiterpene synthase (AsSS4) full-length open reading frame (ORF) cDNA was cloned from wounded stems of Aquilaria sinensis by RT-PCR method. The result showed that the ORF of AsSS4 was 1,698 bp encoding 565 amino acids. Prokaryotic expression vector pET28a-AsSS4 was constructed and transformed into E. coli BL21 (DE3) pLysS. Recombinant AsSS4 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD. Enzymatic reactions using farnesyl pyrophosphate showed that recombinant AsSS4 protein purified by Ni-agarose gel yielded five sesquiterpene compounds, cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This paper reported the first cloning and functional characterization of AsSS4 gene from A. sinensis, which will establish a foundation for future studies on the molecular mechanisms of wound-induce agarwood formation in A. sinensis
