Transcriptome Analysis Of Different Chemical Types Of Perilla Frutescens And Functional Identification Of Terpene Synthase Genes

Objective: To analyze and identify the volatile chemical components in two different chemotypes of Perilla frutescens(L.)Britt.,and compare the differences in the types and relative contents of volatile terpenoids between the two types of P.frutescens.Based on transcriptome analysis of the perillaldehyde type(PA)and perillaketone type(PK)of P.frutescens,the relative expressional profiles of terpene synthase(TPS)gene was analyzed and functional identified in order to study the biosynthesis mechanism of volatile terpene compounds in P.frutescens and explore the correlation between the function of TPS genes and the formation of chemical types of P.frutescens.Methods: The fresh leaves of P.frutescens were collected from AG12(PA Type)and M2(PK Type)plants.The volatile components were extracted by solid-phase microextraction(SPME)method,and were separated and identified by gas chromatography-mass spectrometry(GC-MS).The compounds detected by GC-MS were characterized by searching against NIST 20 mass database.Based on the combination of RNA-Seq and Iso-Seq,the transcriptome dataset of the two chemotypes of P.frutescens were obtained.The transcript sequences were assembled and annotated to obtain a qualified transcriptome data.Combined with the genome data of P.frutescens,we carried out TPS gene screening,sequence correction,relative expression profile analysis,gene cloning and sequence determination from the transcriptome dataset.Finally,the candidate genes with correct sequence were heterologously expressed in Escherichia coli,and the catalytic products were identified by solid phase microextraction combined with GC-MS to clarify the gene function.Results: The volatile terpenoids in PA and PK type P.frutescens were identified by GC-MS.A total of 18 volatile terpenoids were identified in PA type P.frutescens,including9 monoterpenes and 9 sesquiterpenes.PA(29.26%)was the most abundant compound in PA type P.frutescens,followed by caryophyllene(17.39%),α-bergamotene(10.01%),perillyl alcohol(9.87%)and limonene(6.97%),while other compounds containing less than 3%.By comparison,nine compounds were identified in PK-type P.frutescens,of which the highest content was PK(58.62%),with three compounds containing more than10%,namely caryophyllene(15.03%),nerolidol(13.11%)and α-bergamotene(10.66%),and the remaining 5 compounds were less than 3%.A total of 32 PfTPS genes were annotated from the transcriptome data of the two chemotypes of P.frutescens(AG12 and M2).Differential gene expression analysis of these PfTPS revealed that nine TPS genes were highly expressed in the two chemical types of P.frutescens,of which PfTPS3 and PfTPS32 were highly expressed in AG12(PA type),while PfTPS19 was highly expressed in M2(PK type).The expression of six genes in PK type was higher than that in PA type,and the expression of PfTPS1 and PfTPS22 in PK type was significantly higher than that in PA type.The expression of the remaining 17 PfTPS genes in the two chemical types was negligible in P.frutescens.The multiple sequence alignment analysis revealed that 24 PfTPS contained the motif "DDXXD",3 PfTPS contained the motif "DXDD".The phylogenetic tree analysis showed that PfTPS1 and PfTPS4 had the highest homology with the linalool synthase of Salvia officinalis,whereas PfTPS2 and PfTPS3 had the highest homology with the linalool synthase which was characterized in P.frutescens.PfTPS5 was clustered with the myrence synthase of P.frutescens,and PfTPS6 was clustered with germacrenes D synthase.A total of 8 TPS were cloned and named A-PfTPS1,A-PfTPS2,A-PfTPS3,M-PfTPS3,A-PfTPS4,A-PfTPS5,A-PfTPS6,M-PfTPS6 in this study.All sequences had complete open reading frames(ORF),proteins in the length range of 528-607 aa,with theoretical isoelectric points of 61.418-71.112,and all proteins are hydrophilic proteins.Based on gene amplification,sequencing and sequence alignment analysis,a total of 8PfTPS genes were cloned and obtained from the two types of P.frutescens,namely A-PfTPS1,A-PfTPS2,A-PfTPS3,M-PfTPS3,A-PfTPS4,A-PfTPS5,A-PfTPS6,M-PfTPS6,wherein A-PfTPS represents the gene cloned from AG12;M-PfTPS represents the gene cloned from M2.The sequence homology of A-PfTPS3 and M-PfTPS3 was up to 95%.All the cloned sequences have complete open reading frames,the length range of coding proteins was 528-607 aa,and the theoretical isoelectric point was 61.418-71.112.All the PfTPS proteins were predicted to be hydrophilic proteins.The eight PfTPS was heterologously expressed in E.coli using the endogenous FPP of E.coli as the substrate,and the products were detected by GC-MS.The results showed that A-PfTPS1,A-PfTPS2,A-PfTPS3 and M-PfTPS3 had catalytic activity.The catalytic products of A-PfTPS1 were linalool and nerol,and the catalytic products of A-PfTPS2,A-PfTPS3 and M-PfTPS3 were linalool.Conclusion: In this study,the volatile terpenes of two chemical types of P.frutescens were isolated and identified,and the expression profile and relative expression difference of TPS genes in the two types of P.frutescens were analyzed based on the transcriptome analysis.Then,the prokaryotic expression system was used to identify the catalytic function of these PfTPS,which laid a foundation for analyzing the mechanism of volatile terpene biosynthesis in P.frutescens,constructing an efficient heterogenous production platform of terpenes,and exploring the function of TPS genes of P.frutescens.