Functional Study Of Microbial Terpenoid Synthase From Selaginella Jiangnan

Terpenoids are structurally diverse plant secondary metabolites.They not only are essential for plant growth and development but also represent an important role in the various interactions of plants with the environment.Terpene synthase(TPS)is a key that catalyzes the synthesis of terpenoid.Microbial terpene synthase-like(MTPSL)is a new type of terpene synthase,which discovered from Selaginella moellendorffii.MTPSL is evolutionarily more closely related to microbial TPS than to typical plant TPS.MTPSL is widely distributed in nonseed plants but absents from seed plants and green algae.MTPSL contains only an ?-domain and two uncanonical forms:DDxxxD and DDxxx.Meanwhile,they have multiple catalytic functions,like typical plant TPS.At present,SmMTPSL genes are lack of comprehensive identification and functional characterization,however.The study selected S.moellendorffii as the research object.There were five aspects have been researched systematically,which are the effects of different stresses on the release terpenoids from S.moellendorffii,identification,analysis,function prediction of SmMTPSL,enzyme activity detection,gene expression analysis,and antibacterial function research of SmMTPSL.The results are as follows:Selaginella Jiangnanensis was treated with different hormones,and the volatile terpenoids released by it were determined.(1)The S.moellendorffii was treated with different hormones which are methyl jasmonate(MeJA),alamethicin(ALA),6-BA(6-Benzylaminopurine),abscisic acid(ABA),and strigolactone(SL).The volatile terpenoids released from the S.moellendorffii were determined.The results of GC-MS showed that 3,4,1,3,and 2 volatile terpenoids had been detected,respectively.Among them,ALA had the most significant effect on S.moellendorffii.It is indicated that the S.moellendorffii could release different volatile terpenoids in response to different stresses.(2)The third-generation high-throughput sequencing technology was used to sequence the transcriptome of S.moellendorffii.According to bioinformatics methods,8 SmMTPSL genes had been identificatied,analyzed,and functionally predicted.Using TargetP to predict the location of SmMTPSL,the results showed that SmMTPSL46 contains targeting peptides and is located in mitochondria;SmMTPSL5,SmMTPSL 15,SmMTPSL47,SmMTPSL33,and SmMTPSL37 contain signal peptides and are located in the secretory pathway;SmMTPSL19 and SmMTPSL23 are located in the cytoplasm.The results of multiple sequences alignment displayed that the C-terminus of the 8 SmMTPSL contained two highly conserved aspartic acid-rich motifs:DDxx[D/E]and NSE/DTE.Phylogenetic analysis found that SmMTPSL has a closer evolutionary relationship with four ferns MTPSL(AcMTPSL1,CpMTPSL1,Mp1MTPSL,and DbMTPSL).The results of protein three-dimensional structure modeling and molecular docking demonstrated that 8 SmMTPSL structure models could interact with the ligands FPP and GPP to form stable complexes through hydrogen bonds,?-alkyl groups,?-anions,and van der Waals forces.(3)8 SmMTPSL were prokaryotic expressed,and their terpene synthase activities were identified using GPP and FPP as substrates.The results of GC-MS showed that when GPP was as the substrate,the main product of 8 SmMTPSL all were geraniol;when FPP as the substrate,more SmMTPSL could produced ?-farnesene,germacrene,farnesol,and aromadendrene.It was proved that they have monoterpene and sesquiterpene synthase activities and they are multifunctional enzymes.(4)Quantitative real-time PCR was used to detect the expression patterns of SmMTPSL23,SmMTPSL33,and SmMTPSL37 genes after ALA and MeJA treatments.The results suggested that they could respond to the stress of ALA and MeJA,their expression all change to varying degrees,and there are time differences.(5)Based on the antibacterial experiment,the antibacterial activities of SmMTPSL23,SmMTPSL33,and SmMTPSL37 were tested in vitro.The results indicated that the enzymatic products of SmMTPSL23,SmMTPSL33,and SmMTPSL37 have different of antibacterial activity to resist against Pseudomonas syringae pv.tomato DC3000 and Staphylococcus aureus.SmMTPSL23 had the strongest antibacterial activity,followed by SmMTPSL37,SmMTPSL33 has the weakest antibacterial activity.At the same time,it was showed that they all showed a stronger inhibitory effect on the growth of S.aureus compared with Pst DC3000.