Terpene synthase producing patchoulol and elemol, and preferably also pogostol
A technology for patchouliol and patchouliol is applied in the field of preparing patchouliol synthase and can solve the problems of unknown synthase and the like
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Embodiment 1
[0138] GC-MS Analysis of Nardostachys jatamansi
[0139] Nardon pine plants approximately 20 cm tall were purchased from Dingwall IV7 8LX, Ross & Cromarty, Scotland (Poyntzfield Herb Nursery, Black Isle). Plants were cut into leaf material and root material. Weigh 0.5 g of plant material in a pre-cooled glass tube and add 2 mL of dichloromethane. The suspension was vortexed for 1 min, sonicated in an ultrasonic bath for 5 min, and centrifuged at 1500 g for 5 min at room temperature. The supernatant was collected and filtered over a 1 g sodium sulfate column. About 2 microliters were analyzed by GC / MS using gas chromatography as described in detail by Cankar et al. (2015). Patchouli alcohol was identified by comparison with the retention time and mass spectrum of patchouli oil 5 (Sigma-Aldrich).
[0140] result:
[0141] The root of Pinus sylvestris was shown to contain compounds corresponding to patchouli alcohol (retention time 16.4 minutes) and alpha-patchoulene (retent...
Embodiment 2
[0143] RNA extraction and analysis
[0144] RNA was isolated from root material of N. barbatus as follows: about 15 mL of 20 extraction buffer (2% cetyltrimethylammonium bromide, 2% polyvinylpyrrolidone K 30, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0M NaCl, 0.5g / L spermidine and 2% β-mercaptoethanol) were heated to 65°C, then 3g ground tissue was added and mixed. The mixture was extracted twice with an equal volume of chloroform:isoamyl alcohol (1:24), then a quarter volume of 10M LiCl was added to the supernatant and mixed. RNA was precipitated overnight at 4°C and harvested by centrifugation at 10,000 g for 20 min. The pellet was dissolved in 500 microliters of SSTE [1.0 M NaCl, 0.5% SDS, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)] and extracted once with an equal volume of chloroform:isoamyl alcohol. Add two volumes of ethanol to the supernatant, incubate at -20 °C for at least 2 h, centrifuge at 13000 g and remove the supernatant. The pellet was air dried and resuspend...
Embodiment 3
[0146] Example 3: Cloning of Patchouli Alcohol Synthase (NjPAT) of N.
[0147] The full-length open reading frame was amplified from the cDNA of N. The forward and reverse primers shown in Table 1 were designed and the total open reading frame was amplified by fusing the reading frame to the C-terminus of the His-6 tag in plasmid pCDF-DUET-1 (Novagen). A total of 5 different terpene synthase ORFs were cloned. Using primers 27692-Fw (SEQ ID NO: 1) and 27692-Re (SEQ ID NO: 2), two different closely related cDNAs were cloned. One of these proteins (pTS11-1 ) encoded a protein lacking 70 amino acids relative to the protein encoded by contig 27692 above. Another clone (pTS11-2) included the cDNA sequence (SEQ ID NO:3) encoding the protein SEQ ID NO:4.
[0148]Cloned variants were analyzed by sequencing the TS insert. Different variants were introduced into chemically active E. coli BL21-RIL (Stratagene) by heat shock transformation and selected on LB agar containing 1% glucose,...
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