Molecular Cloning And Functional Characterization Of DoGGPPs And DoFPPS In Dendrobium Officinale

Dendrobium officinale(Dendrobium officinale Kimuraet Migo)is a rare medicinal plant of famous Dendrobium.Its main medicinal ingredients are polysaccharide and alkaloid,which have the functions of tonifying stomach,nourishing yin and clearing away heat and so on.Previous studies found that alkaloids produced in Dendrobium officinaile probably came from terpenoid metabolic pathways,which include MVA and MEP pathway.Farnesyl pyrophosphate synthase(FPPS)and geranylgeranyl pyrophosphate synthase(GGPPS)is the key enzyme of penyltransferases.In this paper,Do FPPS and Do GGPPS full-length c DNA sequences were obtained through homologous cloning,and related expression analysis was carried out,aiming to study the roles of Do FPPS and Do GGPPS in terpenoid synthesis of Dendrobium officinale,and lay the foundation for further clarifying terpenoid biosynthesis of Dendrobium officinale.The main results are as follows:1.Do FPPS and Do GGPPS were cloned from the genome of Dendrobium officinale by homologous cloning,and the login numbers of Gen Bank were Do GGPPS(KY012247)and Do FPPS(JX679465),respectively.Tissue specificity analysis showed that Do FPPS and Do GGPPS were expressed in all tissues of Dendrobium officinale.2.The protocorm of Dendrobium officinale was treated with different hormones and inducers,and the expression levels of Do FPPS and Do GGPPS were detected by q RT-PCR.The results showed that methyl jasmonate had a induction effect on Do FPPS and Do GGPPS in D.officinale,and the induced expression level showed a trend of increasing first and then decreasing.SA only had a induction effect on Do GGPPS.The trend of induction was increasing first and then dropping.ABA had a induction effect on Do FPPS and Do GGPPS in D.officinale,and the induction tendency showed increasing first and then falling.The non-biological inducer Ag+ had a induction effect on Do FPPS and Do GGPPS in D.officinale.The induced trends all showed a trend of rising first and then decreasing.3.After the construction of eukaryotic expression vector,the subcellular localization and the complementation test were done.Do FPPS and Do GGPPS were located in the chloroplast by subcellular localization analysis.And the transformation of Do FPPS and Do GGPPS into Arabidopsis thaliana was induced by agrobacterium mediated methods.q RT-PCR analysis showed that Do FPPS and Do GGPPS could influence the expression levels of downstream terpenoid synthase genes in overexpressing transgenic Arabidopsis thaliana lines.4.The interference expression vectors p CAMBIA1301a-Do FPPS and p CAMBIA1301a-Do GGPPS were successfully constructed.Tobacco leaves were transformed by agrobacterium tumefaciens and the expression levels of Do FPPS and Do GGPPS in tobacco were detected by transient expression analysis.It was shown that the transformation of Do FPPS and Do GGPPS RNAi fragments respectively could reduce the expression levels of Do FPPS and Do GGPPS in the tobacco leaves of the treatment group,and demonstrated that the RNAi interference carrier played a role.