Screening And Functional Analysis Of The TPS Gene Of Sesquiterpene Synthetase From Chrysanthemum Aromaticum

Chrysanthemum aromaticum is a rare herb from Shennongjia area.It has golden flowers,small thin stems,and extremely tulip leaves.It can be widely used in medicine,spice industry and daily chemical industry with high economic value after deep processing and full development of volatile oil components.Terpenoids are important components of aromatic substances and are produced by a variety of enzymes.Therefore,it is important to clarify the regulatory mechanism of terpene synthase on the synthesis of aromatic compounds for the development of new ornamental chrysanthemum cultivars by exploiting the aromatic traits of Chrysanthemum aromaticum.In this paper,the function of sesquiterpene synthase,an important branch of terpene synthase,was studied by transcriptome bioinformatics analysis,gene cloning,real-time fluorescence quantitative PCR,GC-MS detection,and other methods,which provided some theoretical basis for further exploring the function of terpene synthase in Chrysanthemum aromaticum.The main test results of this paper are as follows:(1)Bioinformatics method was used to analyze the transcriptomic sequencing results of three different chrysanthemum leaves: Chrysanthemum aromaticum,Chrysanthemum indicum and Chrysanthemum nankingense.38 TPS-a family genes were obtained.Five sesquiterpene family genes were screened with higher expression levels in Chrysanthemum aromaticum leaves than Chrysanthemum indicum and Chrysanthemum nankingense as screening criteria:TPS-a1,TPS-a2,TPS-a3,TPS-a4,TPS-a5.Five sesquiterpene synthase genes were successfully cloned using c DNA as template.The sequence lengths were 1823 bp,1824 bp,1801 bp,1768 bp and 1702 bp,respectively.Their domains,homology and phylogeny were analyzed.It is speculated that it is related to the synthesis of aroma substances in Chrysanthemum aromaticum.(2)Different leaf positions and tissues of Chrysanthemum aromaticum were sampled,and real-time quantitative PCR was carried out using c DNA as template.The results of real-time fluorescence quantitative PCR using c DNA template showed that TPSs gene was expressed in different tissues and organs,and TPS-a1 and TPS-a2 were the highest in the 9th and 10 th leaves.The expression levels of TPS-a3 and TPS-a4 in mature leaves were higher than those in young leaves.The expression level of TPS-a5 was higher in leaves 2-6,but lower in young and old leaves.The expression level of TPS-a1 was the highest in flowers,TPS-a2 was the highest in stems,TPS-a3 was higher in stems and buds,there was no significant difference in the expression of TPS-a4 in different tissues and organs,and the expression level of TPS-a5 in leaves was much higher than that in other parts.At the same time,real-time quantitative analysis of TPSs expression in the brain leaves of Chrysanthemum aromaticum,Chrysanthemum indicum and Chrysanthemum nankingense showed that TPS-a1-5 gene expression in the leaves of Chrysanthemum indicum and Chrysanthemum nankingense was very low or none,which was consistent with the transcriptome data.(3)Five expression vectors of TPS-a family genes were constructed and transferred into yeast strains.Yeast cells were cultured at 28 ℃,and the yeast metabolites were extracted 2-3days later.GC-MS detection results showed that the sesquiterpene gemalene A was detected in the metabolites transferred into TPS-a1,a2 and a4 genes.The sesquiterpene β-caryophylene was detected in yeast metabolites transferred to TPS-a3 and a5 genes,and the yield was higher.Combined with the sesquiterpene synthesis pathway,it is indicated that the TPS-a enzyme can catalyse the production of sesquiterpene compounds in the presence of substrate FPP.It is further indicated that the sesquiterpene synthase TPS-a gene plays an important role in the production pathway of aroma substances and can regulate the synthesis of sesquiterpene compounds.