Cloning And Metabolic Engineering Application Of Perilla Frutescens Synthase Gen

Volatile oil is an important secondary metabolite in plants,and it is also the main medicinal component of aromatic traditional Chinese medicine.Perilla volatile oil is the main functional component of perilla leaves,which has excellent antioxidant,antiaging,immune system enhancement,antibacterial and anti-inflammatory,and tumor growth inhibition effects.There are abundant types of monoterpenes in perilla leaves,and perilla can be divided into different chemical types according to the main types of monoterpenes.Monoterpene compounds are mainly formed by the catalysis of monoterpene synthase(Monoterpene synthase).Monoterpene synthases are a large gene family.Although there have been a few reports on the catalytic functions of perilla monoterpene synthases,systematic studies are still lacking.Identifying the catalytic function of perilla monoterpene synthase and using metabolic engineering to produce monoterpenes is of great significance for the large-scale production of important monoterpenes.Therefore,this research group collected 43 Perilla frutescens as materials to evaluate the types of volatile oils of different perilla germplasms and the antibacterial activities of different types of volatile oils.Based on the previous four chemotype transcriptomes and co-expression analysis,four monoterpene synthase encoding genes were screened and cloned,and the catalytic products of monoterpene synthase were identified by prokaryotic expression in vivo and enzyme activity in vitro.The specific research results are as follows:1.Analysis of volatile oil content of perilla germplasm resources.18 leaf-type leaves and Twenty-five oil-type of Perilla frutescens at the seedling stage were taken,and the volatile oil components were detected by GC-MS to identify the volatile oil types.It was found that 11 perillaldehyde(PA,perillaldehyde)varieties(61%)were detected in leaf-type perilla,including PK,PL,PT and other chemical types.Perilla aldehyde type perilla is a variety of perilla specified in the pharmacopoeia,and the screening material is further utilized as a medicinal perilla.Oil-type perilla species include 8 perillaketone(PK,perillaketone)(32%),8 perillene(PL,Perillene)(32%),and chemical types such as PA,PP,and PPA..2.Evaluation of the antibacterial effect of perilla volatile oil.Based on the perilla aldehyde-type and perillene-type volatile oils with more functional activity reports,the antibacterial effects of Escherichia coli,Salmonella,and Staphylococcus aureus were evaluated respectively.The results showed that the two kinds of perilla volatile oils have obvious inhibitory effect on the tested strains,especially the bacteriostatic effect of perillene on Salmonella,and the bacteriostatic effect at 100%concentration is higher than that of the positive control,which can reach 115% of the control.The good bacteriostatic effect of the two perilla volatile oils provides theoretical support for the development of perilla bacteriostatic functional products.3.Perilla TPS gene cloning and bioinformatics analysis.Based on the previous four chemotype transcriptomes and co-expression analysis,four important TPSencoding genes were identified,and cloned in the same chemotype material in this study.Specific cloned genes include Pf TPS28,Pf TPS29,Pf TPS85,and Pf TPS86,encoding 598,594,605,and 598 amino acids,respectively.Both contain two conserved domains of terpenoid synthases,the TPSN/C-terminal conserved domain.Cloning and bioinformatics analysis of TPS gene in Perilla frutescens.Multiple sequence alignment and phylogenetic tree analysis showed that TPS genes encoding monoterpene synthases could be grouped into three main groups.Among them,the protein encoded by Pf TPS86 has the closest clustering relationship with the reported perilla geraniol synthase,and the homology of their encoded proteins is 100%.The proteins encoded by Pf TPS28,Pf TPS29,and Pf TPS85 have the closest clustering relationship with the reported perilla linalool synthase,and the identities of their encoded proteins are 74%,73%,and 100%.4.Prokaryotic expression in E.coli to verify the function of the candidate TPS gene.The prokaryotic expression vector p ETDuet-1 was used to clone and obtain four TPS genes to construct recombinant plasmids respectively.Escherichia coli containing recombinant plasmids were detected by gas chromatography-mass spectrometry after fermentation and culture,bacterial liquid lysis,and product enrichment.The results showed that the main catalytic product of Pf TPS29,Pf TPS28 and Pf TPS85 was linalool,the secondary catalytic product was citronellol,and the catalytic products of Pf TPS86 were citronellol,and geraniol.5.In vitro enzyme activity to verify the function of TPS.On the basis of prokaryotic expression in E.coli to verify the function of candidate TPS genes,the four genes were respectively constructed into prokaryotic expression vector p ET32 a recombinant plasmids,which were incubated and expressed in E.coli,and the recombinant proteins were isolated for enzyme activity identification.It was found that Pf TPS28,Pf TPS29 and Pf TPS85 catalyzed the production of linalool when GPP was used as the substrate.Pf TPS86 catalyzes the production of geraniol,which is consistent with the results of prokaryotic expression in E.coli.In this study,43 perilla germplasm volatile oil types and two types of chemotypes were evaluated for bacteriostatic activity.Four monoterpene synthaseencoding genes were cloned,and two types were identified by prokaryotic expression in vivo and enzyme activity in vitro: important TPS catalyzing the production of linalool and geraniol.The research results provide a research basis for the development and utilization of volatile oil of perilla germplasm resources and the metabolic engineering utilization of perilla monoterpene synthase.