The Full Length CDNA Cloning And Functional Analysis Of DoTPS13 And DoTPS14 From Dendrobium Officinale

Dendrobium officinale Kimura et Migo belongs to the Dendrobium genus,which is a kind of perennial herb.It is a rare Chinese medicinal material which has the functions of tonifying stomach and promoting fluid,nourishing yin and clearing heat.Terpenoids are important secondary metabolites with the important medicinal functions.Although we all know that terpene synthetase is the key enzyme of the terpene synthesis pathway,which directly catalyzes the production of corresponding terpenoids from different precursors.While,the detection methods of volatile terpenes and the function of terpene synthetase in D.officinale are still not clear.In this study,HS-SPME-GC-MS was used to analyze the volatile terpenes in different tissues from D.officinale,then two terpene synthase genes were cloned and their functions were verified,which help to analyze the functions of terpene synthase in the terpene synthesis pathway of D.officinale.The main results are as follows:（1）HS-SPME-GC-MS was used to detect the volatile terpenes in cultured seedlings,flowers and protocorms of D.officinale.The results showed that 6 and 10 sesquiterpenes were detected in culture seedlings and protocorms,respectively.And 7 monoterpenes and 4sesquiterpenes were detected in flowers.Monoterpenes only exist in flowers,the species and contents of terpenes are different from different tissues.（2）The full-length c DNA sequences of DoTPS13 and DoTPS14 were cloned.The full-length c DNA of DoTPS13 was 1665 bp,encoding 554 amino acids with Gen Bank accession number MT512058,while the full-length of DoTPS14 was 1653 bp,encoding550 amino acids with Gen Bank accession number MT512059.Tissue-specific analysis showed that DoTPS13 and DoTPS14 were expressed in all tissues,and that DoTPS13 was highly expressed in the protocorm and DoTPS14 was highly expressed in the stem.After treatment with Me JA,SA and ABA,the expression levels of DoTPS13 and DoTPS14 were up-regulated.（3）The prokaryotic expression vectors p ET32a-DoTPS13 and p ET32a-DoTPS14 were constructed,and DoTPS13 catalyzed FPP to produceγ-Murrolene andβ-Guaiene by in vitro enzyme activity detection.DoTPS14 catalyzed FPP to produceα-Elemene,γ-Elemene,α-Himachalene,γ-Himachalene.Among them,the main productγ-Murrolene of DoTPS13 was detected from flowers in D.officinale,and the main productα-Elemene of DoTPS14 was detected from protocorms in D.officinale.（4）The eukaryotic expression vectors p CAMBIA1304-DoTPS13 and p CAMBIA1304-DoTPS14 were constructed,through agrobacterium-mediated transformation of arabidopsis thaliana,we screened to obtain T₃generation overexpressed transgenic arabidopsis thaliana.According to the stress resistance test of DoTPS13 and DoTPS14transgenic arabidopsis thaliana,the root length of transgenic lines was longer than wild type,suggesting that DoTPS13 and DoTPS14 might be related to stress resistance.Analysis of the expression levels of genes related to terpene synthesis in overexpressed arabidopsis thaliana showed that the expression levels of HMGR and FPS genes upstream of terpene synthesis were significantly up-regulated.