Cloning And Functional Analysis Of Patchoulol Synthase Gene Responsible For Sesquiterpenes Biosynthesis In Pogostemon Cablin

Plant sesquiterpenes are secondary metabolites with enormous structural diversity and show various biological activities,which play significant roles in plant growth and survival.Given the commercial and ecological importance of sesquiterpenes,metabolic engineering of sesquiterpenes,based on genetic engineering,has become one of the most promising approaches for increasing target sesquiterpenes in plant.One of the important traditional medical materials,Pogostemon cablin(Blanco)Benth.is a perennial herb in the genus Pogostemon of the Lamiaceae family.Generally,its dry stem and leaf are both medical parts for clinical use.Research into the phytochemical constituents and pharmacological activities shows that oil accumulated by P.cablin possesses antioxidant,analgesic,anti-inflammatory,antibacterial,anti-fungal,antithrombotic,antidepressant and many other activities.Patchouli oil is unique because it consists of diverse sesquiterpenes,rather than a blend of different mono-,sesqui-and di-terpene compounds.Patchoulol synthase(PTS)is a putative key enzyme of sesquiterpenoids biosynthesis in patchouli,catalyzing the conversion of farnesyl diphosphate(FDP)to patchoulol as the major product,producing at least 13 additional sesquiterpenes.In this work,molecular cloning,identification and functional characterization of PTS were carried out by using RACE,construction of expression plasmids and q PCR technologies.The main findings were as below:1.Full length c DNA sequence of PTS gene was separated from patchouli leaf for the first time.This full-length c DNA was 1939 bp,containing a 1659 bp open reading frame(ORF)coding for 552 amino acids,a 61 bp 5?-untranslated end,and a 219 bp 3?untranslated sequence,and the sequence deposited in Gen Bank(Accession No: MG386648).2.The p ET32 a was chosen as the expression vector of PTS gene to construct recombinant plasmid and expressed in Escherichia coli.The recombinant enzyme was identified as patchoulol synthase by Western blotting and LC-MS,and designated as Pc-PTS1.And the recombinant plasmid had a 1713 bp ORF coding for 570 amino acids,which had a theoretical molecular weight of 65.77 k Da and an isoelectric point of 5.44.The amino acid sequence homology between Pc-PTS1 and other terpene synthases varied(45% – 63% identity).Pc-PTS1 also had 6 highly conserved regions of plant terpene synthases and 6 additional regions with high conservation of angiosperms sesquiterpene synthases.The analysis of transmembrane domains showed that Pc-PTS1 was not a signal peptide,suggesting that Pc-PTS1 was cytoplasmic enzyme.Prediction of secondary structure revealed that ?-helix and random coil were the main structural conformations in Pc-PTS1 protein.3-D structure modeling showed that 3-D structure of Pc-PTS1 was similar with(+)-?-cadinene synthase isozyme XC1 from Gossypium arboreum.3.Obvious spatiotemporal expression difference was found in different P.cablin cultivars.Expression analyses in both HN-1 and HN-2 cultivars showed that peak expression for leaves and stems occurred in November.The spatiotemporal expression profile for PTS in SP-1 and SP-2 cultivars showed that peak PTS transcription of leaves occurred in August.Similar to the results from the leaves of the HN-1 and HN-2 cultivars,PTS in stems of the SP-1 cultivar was abundantly expressed in November,whereas the highest PTS expression in stems of the SP-2 cultivar was observed in September.Over the course of leaf development in cultivars YN-1 and YN-2,PTS transcript levels appeared to be relatively constant from July to October,with peak expression levels occurring in November.The PTS gene expression profile for stems of cultivar YN-1 peaked in October,while PTS for stems of the cultivar YN-2 was highly expressed in November.Significant levels of PTS expression were detected in November in both leaves and stems of cultivar GY.4.Correlation analyses between PTS transcript abundance and volatile constituents in the seven P.cablin cultivars revealed that no statistically significant correlation coefficient could be detected between PTS expression and either patchoulol concentration in leaves,pogostone concentration in leaves,patchoulol concentration in stems,and pogostone concentration in stems from a range of seven patchouli cultivars.5.It is observed that the oscillations of PTS transcript persisted in P.cablin during the daily cycle,with peak expression occurring at 1:00 h,5:00 h,7:00 h,9:00 h,16:00 h,19:00 h and 22:00 h.Generally,PTS had lower oscillation frequency and transcription abundance at daytime than night.In conclusion,the cloning and characterizations of the PTS gene will provide a potential guide in further investigation on mechanism of sesquiterpene biosynthesis,as well as producing highly value sesquiterpene at large scale by biotechnology.This work will not only be beneficial to the patchouli resources conservation but also provide a basis for the full development and utilization of patchouli resources.