Function Analysis Of Specific Genes Of Terpene Synthase(TPS)in Jasmine

Jasmine [Jasminum sambac(L.)Aiton] is a plant of the genus Jasminum in the family Oleaceae and its aroma is an important indicator of its economic and horticultural value as a fragrant flowering plant.Linalool and α-farnesene are the characteristic aromatic substances of jasmine flowers and are monoterpenes and sesquiterpenes,respectively,synthesised by the MEP(methylerythriol phosphate)and MVA(mevalonic acid)terpene synthesis pathways.In both pathways,Terpene synthase(TPS)is an important late synthase that determines the type of terpene.To investigate the TPS for the synthesis of linalool and α-farnesene in jasmine,this study screened and carried out a series of studies based on the jasmine flower and leaf transcriptomes,and the main findings are as follows:(1)Four TPS candidate genes were screened based on jasmine flower and leaf transcriptomes.Their expression patterns during flower opening were synchronized with the release of linalool and α-farnesene and their expression levels were high.JsTPS01 belongs to the TPS-a subfamily,JsTPS02 and JsTPS03 belong to the TPS-b subfamily and JsTPS04 belongs to the TPS-b subfamily.JsTPS04 belongs to the TPS-e/f subfamily.The results of multiple sequence alignment showed that JsTPS01 and JsTPS03 contained aspartic acid-rich DDXXDD and NES/DTE sequences with the arginine-tryptophan motif R(R)X8W at the N-terminus;JsTPS02 contained DDXXDD and NES/DTE sequences with the motif R(R)X8W at the N-terminus,possibly containing a plastid transit peptide of length 25 aa;JsTPS04 contains DDXXDD and NES/DTE sequences.(2)The subcellular localization of the four TPS genes was analysed using jasmine petal protoplasts as well as Arabidopsis leaf protoplasts.The results showed that JsTPS01 and JsTPS03 were localized in the cytoplasm;JsTPS02 was localized in the plastid;JsTPS04 was localized in the cytoplasm in Arabidopsis protoplasts and in the plastid in jasmine protoplasts.(3)Heterologous transformation of JsTPS01 into Saccharomyces cerevisiae resulted in the synthesis of the sesquiterpene α-cadinol(C15H26O).Four TPS genes were constructed into the yeast expression vector pDRTxa and transformed into Saccharomyces cerevisiae By4742 strain,in which JsTPS01 transformed secretion of Saccharomyces cerevisiae showed an additional sesquiterpene α-cadinol compared to the control.(4)Transient transformation of tobacco(Nicotiana benthamiana)by JsTPS04 resulted in the synthesis of the monoterpene linalool.Four TPS were constructed into the plant expression vector pK7FWG2.0 and transiently transformed into tobacco to detect the volatile substances.(5)The recombinant proteins JsTPS02 and JsTPS04 were obtained by heterologous expression in E.coli and purification.The four TPS genes were constructed into the prokaryotic expression vector pET21HA,transformed into Rosetta(DE3),and expression was induced at OD600=0.6-0.8,1 m M IPTG,20℃ overnight,and purified using a Ni-NTA column.The results showed that the proteins expressed by JsTPS01 and JsTPS03 in E.coli were mainly in the form of inclusion bodies and could not be purified.JsTPS02 and JsTPS04 were successfully expressed and purified proteins were obtained.(6)JsTPS02 and JsTPS04 catalyzed the in vitro reaction of GPP to produce linalool,and JsTPS04 catalyzed the reaction of FPP to produce trans-nerolidol.The purified JsTPS02 and JsTPS04 recombinant proteins were reacted with different concentrations of GPP,FPP and GGPP substrates in specific buffers,and the reaction products were determined by GC-MS and the enzymatic kinetic parameters Km(substrate concentration at half the maximum reaction rate)and Vmax(maximum reaction rate of the enzyme)were calculated.The results showed that JsTPS02 and JsTPS04 could catalyse the reaction of GPP to produce linalool with Km values of 28.180 μM and 2.511 μM and Vmax values of 47.680 ng/μg protein·h and 6.202ng/μg protein·h,respectively.The reaction of JsTPS04 with the substrate FPP produced trans-nerolidol with a Km value of 7.577 μM and a Vmax value of 7.572ng/μg protein·h.In summary,four TPS genes,JsTPS01,JsTPS02,JsTPS03 and JsTPS04,which are highly expressed in flowers and synchronized with the release of terpene aroma,were screened based on the transcriptome of jasmine flowers and leaves,among which JsTPS01 is a sesquiterpene synthase gene,JsTPS02 is a linalool synthase gene and JsTPS04 has the dual function of synthesizing monoterpene/sesquiterpene.The results of this study can provide a basis for the molecular breeding of jasmine high-perfume germplasm based on TPS genes.