Catalytic Function,Spatiotemporal Expression And Regulation Mechanisms Of CsNES In Camellia Sinensis

Aroma quality is considered as the key factors for high flavor quality of tea.Volatile terpenoids are the major tea aromatic components and important for aroma quality.Recent studies revealed that transcription factor MYC2 controls the relevant terpenoid synthase genes expression through jasmonic acid(JA)signaling pathway.Volatile sesquiterpenoids function in defense,stress tolerance,and signaling transduction between and within plants.Nerolidol,a floral odor sesquiterpene,usually stored as glycosidically bound form and serves as aroma precursor in tea leaves,thus is important for tea aroma quality.Thus,it's meaningful to uncover the mechanisms of sesquiterpenoids(nerolidol)biosynthesis,regulation and storage.This study aims to explore the function,spatiotemporal expression and regulation mechanisms of a nerolidol synthase gene and to promote the tea aroma quality and tea resistance.Partial sequences of farnesyl pyrophosphate synthase gene(CsFPS),nerolidol synthase gene(CsNES)and MYC2 gene were found from RNA-seq transriptone sequence data.Fresh tea leaves [Camellia sinensis(L.)O.Kuntze cv.Nongkangzao] were used as the test materials.Full length of CsFPS,CsNES and MYC2 were cloned through the RACE-PCR technology.CsNES containing 1659 bp coding sequence,encoding a potein of552 amino acid residues and the molecular weight(MW)is 63.5 KDa,pI=5.46;CsFPS containing 1029 bp coding sequence,encoding a potein of 342 amino acid residues and the MW is 39.5 KDa,pI=5.67.Multiple protein sequence alignment showed that Cs NES has a metal ion binding site“DDXXD” and a terpene synthase activity site “EDXXD”.Prokaryotic expression vector pET32a-CsNES was conducted and expressed in E.coli Rosetta.In vitro assay,detected by HS-SPME,showed that the recombinant protein CsNES catalyzed the conversion of FPP to(E)-nerolidol and farnesene,and GPP to a small amount of linalool.Transiently expression of the CsNES-mGFP5 fusion protein in Allium cepa epidermal cells revealed that GFP ?uorescent signals exclusively presented in cytosol,thus suggesting that CsNES involved in cytosolic(E)-nerolidol biosynthesis.MeJA treatment can induce CsNES expression in tea leaves.Using the Genome Walking Kit,742 bp CsNES promoter sequence upstream of the transcription start site was cloned.cis-element analysis revealed two E-boxes located in-214 and-688 bp using on-line program PLACE,suggesting that CsNES promoter can interact with CsMYC2 and take part in JA signaling pathway.Transgenic tobacco plants over-expressed CsNES,CsFPS was generated for further functional characterization.However,the biofunction analysis has not started since the amount of transgenic plants are not enough now with CsNES is 4 plants and CsFPS only 1plant.In order to verify the biosynthesis and regulation mechanism of tea sesquiterpenoids,the tea Cs NES and its upstream gene CsFPS were cloned.Prokaryotic expression and subcellular localization of CsNES were further done for biological functions identification.Cloning and analysis of the promoter of CsNES provides an important basis for nerolidol biosynthesis and regulation mechanism in tea plant.The results of this study provide theoretical basis for crop resistance and tea aroma quality enhancement.