Screening And Functional Verification Of Tea β-caryophyllenes Synthase Based On The Analysis Of The Terpene Synthase Family

Camellia sinensis L is an important cash crop for horticultural leaves in China.But like other cash crops,tea also faces certain insect attacks.Meanwhile,the particularity of tea as a drink also makes the problem of pesticide residue more prominent.The quality and drinking safety of tea is the focus of people’s attention.The damage of diseases and insect pests in tea plantation is serious,which leads to the reduction of production by 15% ～ 20% every year.Volatile terpene compounds are important aroma active substances in flowers and fruits of plants,and have important physiological effects,participating in the direct or indirect defense of tea.Therefore,the mining of genes related to terpenoid synthesis is of great significance for the green prevention and control of diseases and insect pests of tea tree.Several terpene synthases have been identified in tea,but many more TPSs have not been identified.In order to fully understand the terpene synthase(Cs TPSs)in tea tree,40 Cs TPSs were found by analyzing the tea tree genome sequence.The terpene synthase gene family of plants is divided into 7 phylogenetic subfamilies,including 5 in tea tree.On this basis,this study systematically analyzed the evolutionary relationship of Cs TPS gene family members,the structural characteristics of endons and exons,the conserved structural domains,their expression in different tea tissues and their response to methyl jasmonate.Gene expression analysis showed that the expression of Cs TPSs in buds,leaves and flowers was significantly higher than that in other tissues,which was consistent with the unique flavor of tea.Feeding by tea inchworm can significantly induce the synthesis and release of related terpenoids from Cs TPS27.GC-MS analysis showed that many terpenoids can be synthesized from the leaf of the tea looper chewing tree,including-caryophyllene which has anti-insect effect.while the uneaten leaves had no or only a small amount of agaricocarytene synthesis,suggesting that Cs TPS27 may have a certain function in the anti-insect defense of tea tree.In order to further verify the biological function of these Cs TPSs,a related gene Cs TPS27 with strong response to methyl jasmonate was screened in this study.PCR was used to clone the full length of the gene,and the evolutionary tree analysis showed that Cs TPS27 belonged to the TPS-a branch.The full length of Cs TPS27 was connected to p GEX-4T1 vector and transformed into escherichia coli.The recombinant protein was expressed.Enzyme activity was analyzed in vitro with GPP and FPP as substrates.The results showed that Cs TPS27 could use FPP as the substrate to catalyze the synthesis of monocyclic structures in vitro.Monocyclic products include β-elemene,Germacrene D,γ-elemene,α-muurolene and α-caryophyllene,and double ring β-caryophyllene,γ-muurolen,α-cadinene,γ-cadinene.(+)-aromadenderenee,δ-cadinene.followed by,the third ring structure of α-cubebene and α-copaene.GPP cannot be used as substrate.Cs TPS27 may be a multifunctional sesquiterpene synthase gene The in vivo activity experiments of tea plants may need to be further improved.In summary,based on the systematic analysis of the characteristics of Cs TPSs family of tea tree,this study revealed that Cs TPS27 is a new sesquiterpene cyclase of tea tree.This study not only helps to understand the Cs TPSs family of tea tree,but also provides a theoretical basis for the biosynthesis and regulation of various cyclified sesiterpenes in tea tree.