Determination Of Floral Volatile Components And Prelimin Ary Study On Function Of Two Terpene Synthases In Wintersweet

Wintersweet(Chimonanthus praecox L.),a traditional fragrant plant that originates from China,is a well-known winter-flowering deciduous shrub.The unique floral scent wintersweet made it one of most popular ornamental plants in China.It was extensively cultivated as garden,potted,and cutflower plants.In this paper,the floral scent components of four periods of scent H29 and scentless SW001 accessions in wintersweet were determined by headspace solid-phase microextraction coupled with gas chromatography mass spectrometrometry(HS-SPME-GC-MS)CpTPS5 and CpTPS16,two terpene synthase genes were identified from H29 and SW001 of wintersweet.Expression levels of CpTPS5 and CpTPS16 of different flower stages from wintersweet H29 and SW001 were detected by quantitative real-time PCR(q PCR).In addition,for functional characterization of CpTPS5 and CpTPS16,the coding regions of the two genes were cloned into binary expression vector,and cloned genes were transformed into tobacco.Qualitative and quantitative analyses of floral scent components in transgenic tobacco were performed by HS-SPME-GC-MS,The main results are as follows:1.64 kinds of floral scent components were identified in H29 and SW001,most of which were terpenoids and benzenoids.The total amount of volatile compounds in the profusw flowering period of H29 was 3449.48ng/g,and the higher contents included Linalool,Acetic acid phenylmethyl ester,Methyl salicylatel,respectively,the SW001's total volatiles compounds was 9886.26ng/g at the same time,included: trans-?-Ocimene,Bicyclo(3.1.1)heptane-2,3-diol,2,6,6-trimethyl-,2,6-Dimethyl-1,3,5,7-octatetraene,E,E-,Methyl salicylate,Benzoic acid methyl ester.2.The determination of floral scent components in four periods of two accessions.It showed that the total content of terpenes volatile compounds rised from petal baring stage to the final flowering stage in H29.The variation of monoterpene volatile compounds was the same,however,the total content of sesquiterpene volatile compounds 86.57ng/g appeared in petal baring stage,then declined,a little pick up in final flowering period.In SW001,the total content of terpenes volatile compounds rised from petal baring stage to profuse flowering period and reached its peak at this time,then declined at the final flowering period.The variation of monoterpene volatile compounds was the same,however,the total content of sesquiterpene volatile compounds in petal baring period was more than that in initial flowering period.The 44.46ng/g content of sesquiterpene appeared in profuse flowering period and declined at the final flowering period.3.Protein sequence alignment showed that CpTPS5 and CpTPS16 had no difference in H29 and SW001 accessions.Construction the phylogenetic tree with other terpenes homelogous proteins,the results showed that CpTPS5 was clustered together with TPSa sesquiterpene synthase family,and is similar to Caryophyllene synthetase,while CpTPS16 was clustered together with TPSb monoterpene synthase terpene family.4.CpTPS5,CpTPS16 Real-time PCR results showed: as an up-regulated expression gene in H29,CpTPS5 gene expression increased in initial stage then declined,it is thirteen times as deep as SW001 in profuse flowering period.While CpTPS16 as an up-regulated expression gene in SW001,the expression was higher than H29 during the four periods,the expression trend of CpTPS16 showed same between 2 accessions.Gene expression continuously rised from petal baring stage to the final flowering stage.5.The qualitative and quantitative analysis of the aroma components of CpTPS5 and CpTPS16 in tobacco showed that the trend of monoterpene and sesquiterpene volatile compounds emission was regular.There were no significant volatiles difference between transgenic plants and wildtype tobacco,but the content of volatile terpenes was much higher than that in wildtype.Absolute quantitative calculation of five volatile compounds:Caryophyllene,Linalool,Limonene,1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-Naphthalene,Methyl hexanoate.It was found that the content of the four terpenoids volatile compounds in the transgenic CpTPS16 was about two times as deep as CpTPS5,and the amount of volatile compounds was much higher than that in the wildtype and p CAMBIA2300.However,the emission of chain ester methyl hexanoate in CpTPS5 and CpTPS16 was basically the same,with no significant difference.