Cloning,expression And Functional Analysis Of Moso Bamboo Terpene Synthase Genes(TPSs)

Terpenoids are a class of compounds derived from isoprene as structural unit.They are by far the largest family of plant secondary metabolites,exhibiting a variety of chemical ecology effects.For example,one of the main components of flowery flavour in plants are terpenoids,which are considered as an important signal tolure insect pollination and fertilization.Meanwhile,volatile terpenoids could be released to attract predators to protect plants against insect damage.In addition,terpenoids play an important role in plant-insect and plant-pathogen interaction,and they are reported to involve in plant defense responses directly or indirectly.Moso bamboo(Phyllostachys edulis)is one of the most representative bamboo resourses among gramineous plants,and also the most ecological and valuable species in bambusoideae.Due to its wide distribution,high economic value and abundant processing products,it has been used as a model plant for bambusoideae study by researchers.Recently,bamboo genome has been decoded,but till now there are less reports to explore functions of genes contained in this genome database.Using plant molecule biology technique,this thesis aimed to isolate terpene synthase genes(MoTPS)derived from moso bamboo and obtain the complete gene coding sequences.Both Escherichia coli and Pichia pastoris were applied to perform prokaryotic and eukaryotic expression systems,trying to find out engineering bacteria or yeast for high level expression of MoTPS.The fusion proteins were then separated and purified,following by enzyme analysis in vitro for functional characterization.This study could clarify terpenoids metabolic pathway in moso bamboo as well as the functions of the key enzyme involved,and provide reference and reform targets for the industrial production of terpenoids.The main results were as follows:1.Based on moso bamboo genome database,15 potential terpene synthase genes were screened through KEGG automatic annotation and designated as MoTPS1-15.Phylogenetic tree analysis showed that eight genes(MoTPS1-8)belonged to Tpsa subfamily,encoding sesquiterpene and diterpene synthase.MoTPS10-12/14 belonged to Tpsb subfamily,encoding monoterpene synthase.MoTPS13 belonged to Tpsg subfamily.Yet neither MoTPS9 nor MoTPS15 were members of any known terpene synthase family.Analysis of protein subcellular location indicated that most of them were distributed in cytoplasm while less were located in chloroplast,that was corresponding to MVA and MEP pathway involved in terpene synthesis in plants.After changing annealing temperature by gradient PCR,8 of 15 gene(MoTPS2/3/5/6/11-14)were successfully cloned,using several amplification templates from different moso bamboo tissues.For MoTPS12 or MoTPS13,another transcript formed owing to alternative splicing,renamed as MoTPS12 S and MoTPS13 S respectively.2.There were five expression vectors used for prokaryotic expression,including pGEX4T-1,pET30a(+),pET32a(+),pET28a-sumo and pColdI-sumo.After digested by appropriate restriction enzymes,the ten genes mentioned above were ligated with these vectors respectively.A total of 18 recombinant plasmids were successfully constructed,containing pGEX4T-1-MoTPS2/12/12S/13/13 S,pET30a-MoTPS2,pET32a-MoTPS2/12/12S/13/13 S,pET28a-sumo-MoTPS2/5/6 and pColdI-sumo-MoTPS2/5/6/14.They were then transformed into different E.coli strains for expression and the results revealed 28 positive clones obtained.By inducible expression and optimization,the target proteins were found to be expressed in the form of inclusion body,existing in the precipitation components after ultrasonication.3.Two eukaryotic expression vectors of p PIC9 K and pPICZ?A were applied to produce secreted and active proteins,that is easy to conduct further enzyme assays.In all,nine recombinant plasmids were successfully constructed,including pPIC9K-MoTPS2 and pPICZ?A-MoTPS2/3/5/6/12/12S/13/13S/14.They were subsequently integrated into Pichia pastoris chromosome by electrotransformation,and the corresponding positive clones were selected by PCR.The methanol-inducible expression and optimization were achieved for four recombinants of MoTPS2/5/6/14 and the soluble enzymes were detected in supernatant of yeast culture.For MoTPS2 gene,the recombinant of pPIC9K-MoTPS2 was transformed into GS115 strain for optimal MoTPS2 expression in the MM medium(pH 4.0)with 1% casamino acid added.After 48-hours induction,the target protein can be detected.For the recombinants of pPICZ?A-MoTPS5/6/14,P.pastoris X-33 strain were used as the host strain for optimal expression in the BMMY medium(pH 6.0).SDS-PAGE analysis suggested that MoTPS5 could be detected after 24-hours induction while MoTPS6/14 appeared after 48-hours induction.4.Through the preliminary purification,the soluble enzyme preparations were subjected to enzyme assays in the presence of farnesyl diphosphate(FPP),geranyl diphosphate(GPP)and geranylgeranyl diphosphate(GGPP)as substrates.The SPME-headspace technique were employed to extract volatile terpenes.The enzyme products were determined by GC-MS analysis and compared with the standards.Functional characterization was achieved for three genes(MoTPS2,MoTPS5 and MoTPS6),of which,MoTPS2 and MoTPS6 were catalytically active,capable of converting FPP to two sesquiterpenes,that is,(E,E)-farnesol and(E)-nerolidol.Therefore,they were speculated to encode farnesol synthase and nerolidol synthase individually.For MoTPS5 gene,the enzyme products were identified as a mixture of primary(E,E)-farnesol and(E)-nerolidol,accounting for a multi-product terpene synthase.MoTPS2,MoTPS5 and MoTPS6 all belonged to Tpsa subfamily,encoding 546,541 and 541 amino acids respectively.Their sequences were submitted to NCBI database and the accession numbers were obtained: MoTPS2(KP097716.1),corresponding protein sequence(AJP67536.1);MoTPS5(KP097717.1),protein sequence(AJP67537.1);MoTPS6(KP097715.1),protein sequences(AJP67535.1).During development of mosobamboo,terpenoids serve as important physiological and ecological effect.This thesis is the first report of terpenoid biosynthesis pathway in moso bamboo,and systematic explains the potential terpene synthase genes in moso bamboo,which will benifit to understand terpenoids biosynthesis pathway and lay the foundation.to produce terpenoidsat industrialized level.