Cloning And Characterization Of (E)-?-farnesene Synthase From Matricaria Recutita L.

Matricaria recutita(L.)is a kind of annual herb from Genus Matricaria in Asteraceae,which contains abundant chemical compounds such as sesquiterpenes and flavonoids,and therefore has significant antiinflammatory,antibacterial,anti-oxidation,hypolipidemic,anticoagulation,antineoplastic and spasmolytic properties.(E)-?-farnesene(E?F)is an important sesquiterpene compound widely exists in a number of plants.As the main component of most aphid alarm pheromone,E?F is involved in aphids defensive chemical communication.To illustrate the metabolic regulation of E?F biosynthesis,on the one hand,we described the cloning and functional analysis of the ?FS gene from M.recutita;on the other hand,the terpenoids secondary metabolites in chamomile fresh leaves after spraying with 100 ?M MeJA were identified by GC-MS analysis.This study primarily discussed the regularity of chamomile terpenoids secondary metabolism adjusted by methyl jasmonic acid and provided a theoretical basis for the regulation of sesquiterpene contents from M.recutita through the genetic engineering means.The main results were as follows:1.The full-length cDNA of ?FS gene was cloned from chamomile by RT-PCR and RACE technology and named Mr-?FS.The full-length ?FS cDNA was 2010 bp,containing an open reading frame of 1725 bp that encodes a peptide of 579 amino acids(GenBank accession number: KM586847).The deduced amino acid sequence has a predicted molecular weight of 67 kDa and pI of 5.28.The deduced amino acid sequence exhibits a considerably higher homology to ?FS from Artemisia annua(about 92% identity)than to ?FS from other plants(about 20-40% identity).Phylogenetic tree analysis also indicated that Mr-?FS has the closest relationship with?FS from A.annua.2.Prokaryotic expression vector pET-28a-?FS was constructed successfully,and fusion protein pET-28a-?FS was expressed in E.coli with relative molecular masses of 67 kDa.The recombinant protein was incubated with farnesyl diphosphate(FPP)and the product was analyzed by GC-MS.The Mr-?FS recombinant protein converted FPP to a single sesquiterpene,which was identified as(E)-?-farnesene.3.qRT-PCR showed that the Mr-?FS was ubiquitously expressed in all of the examined tissues,but at different levels.Mr-?FS expression was highest in leaves and lowest in disk florets.The expression level in leaves was about 6.8-fold higher thanthat in disk flowers.In different floral stages,the lowest level of Mr-?FS mRNA expression was found in fully opened period.Expression of Mr-?FS in bud flowers and semi-opened flowers was 1.6-fold and 2.6-fold higher than that in fully opened flowers,respectively.4.Transcription accumulation was obvious in Mr-?FS gene expression under MeJA treatment.Among them,Mr-?FS transcripts were maximally induced by up to11.5-fold at 24 h after MeJA treatment compared with the control sample.GC-MS analysis showed that all the five detected volatile terpenoids ?-ocimeme,(E)-?-farnesene,germacrene D,?-elemene,and ?-farnesene had significant differences(P < 0.05)compared with the control sample after MeJA treatment.