Abstract
Celastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the present study, T. wilfordii squalene synthase (TwSQS) was cloned followed by prokaryotic expression and functional verification. The open reading frame cDNA of TwSQS was 1242 bp encoding 413 amino acids. Bioinformatic and phylogenetic analysis showed that TwSQS had high homology with other plant SQSs. To obtain soluble protein, the truncated TwSQS without the last 28 amino acids of the carboxy terminus was inductively expressed in Escherichia coli Transetta (DE3). The purified protein was detected by SDS-PAGE and Western blot analysis. Squalene was detected in the product of in vitro reactions by gas chromatograph-mass spectrometry, which meant that TwSQS did have catalytic activity. Organ-specific and inducible expression levels of TwSQS were detected by quantitative real-time PCR. The results indicated that TwSQS was highly expressed in roots, followed by the stems and leaves, and was significantly up-regulated upon MeJA treatment. The identification of TwSQS is important for further studies of celastrol biosynthesis in T. wilfordii.
Highlights
Tripterygium wilfordii (Celastraceae) is an important Chinese herbal medicine used for the treatment o f inflammation [1]
The open reading frame (ORF) cDNA of T. wilfordii squalene synthase (TwSQS) was amplified by PCR using primers TwSQS-F
We found that 100 μM methyl jasmonate (MeJA) treatment could significantly increase the Celastrol is a representative molecule of quinone-methide which are deemed as content of secondary metabolites and the expression level of related triterpenoids genes in Tripterygium wilfordii theorder chemotaxonomic markers of Celastraceae
Summary
Tripterygium wilfordii (Celastraceae) is an important Chinese herbal medicine used for the treatment o f inflammation [1]. Many QMTs have already been isolated from Celastraceae, such as celastrol, maitenin, 22-hydroxytingenone and pristimerin [3]. They have similar chemical structures and biological activities. Celastrol is the first active compound isolated from the root bark of T. wilfordii and is one of the major active ingredients of T. wilfordii [4]. As a representative compound of QMTs, Celastrol has been demonstrated to have promising activities including antioxidant [5], anti-inflammatory [6], immunomodulation [7], and anticancer activities [8]. Celastrol has shown promise as an anti-inflammatory compound for arthritis, lupus, amyotrophic lateral sclerosis, and Alzheimer’s disease [6]. Celastrol is Molecules 2018, 23, 269; doi:10.3390/molecules23020269 www.mdpi.com/journal/molecules
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