Study On Construction And Transformation Of SmIPI-RNAi Vector

Tanshinone is a kind of diterpene quinone compound in Salvia miltiorrhiza,which is the main medicinal ingredient of Salvia miltiorrhiza and has extremely important medicinal value in the treatment of cardiovascular and cerebrovascular diseases.In plants,the metabolism of diterpenoids is regulated by a variety of enzymes,of which isoprene pyrophosphate isomerase(IPI)is the main switch of its downstream metabolic pathway,and the gene encoding the enzyme has now become an effective target for metabolic engineering of pentadiene compounds in Escherichia coli.In this study,the interference expression vector of SmIPI1 and SmIPI2 was constructed by Gateway technique,and the genetic transformation was induced by Agrobacterium tumefaciens,and the positive plants of transformation were detected by resistance marker screening,Egfp fluorescence and qRT-PCR.The effects of SmIPI gene expression on the metabolism of primary and secondary metabolites in MEP and MVA were studied by GC-MS and LC-MS,and provide a basic study for the complete analysis of the role of SmIPI in the MEP and MVA pathways.The main results are as follows:1.Specific gene fragments from SmIPI1 and SmIPI2 were reorganized into expression vector pK7WIWG2D(II)by Gateway technology cloning and subcloning methods.The recombinant plasmid was tested by PCR and sequencing of specific primers,indicating that the interference expression vector was constructed successfully,and obtained the SmIPI1 gene interference expression vectors RSi1 A,RSi1B,and SmIPI2 gene interference expression vectors RSi2 A and RSi2 B.2.Using the Agrobacterium tumefaciens-mediated transformation,the regeneration system of SmIPI-RNAi Salvia miltiorrhiza plant was established with leaf disc as explant,and 400 mg/L Cef,1.0 mg/L 6-BA and 0.1 mg/L NAA MS as induction medium,30 mg/L Kan for resistance screening,0.5 mg/L IBA and 0.5 mg/L NAA 1/2MS as rooting medium.The root tip of the transformed plant was strongly green fluorescence under the fluorescence microscope,and the relative expression levels of SmIPI1 and SmIPI2 genes were down-regulated by 64%-82% and 23%-78%,respectively.3.The phytosterols and the diterpenoids in SmIPI1-RNAi,Sm IPI2-RNAi and V0 control line of Salvia miltiorrhiza plant were detected and analyzed by GC-EI-MS/MRM and UPLC-ESI-MS/MRM,respectively.The results showed that the total content of campesterol,stigmasterol and ?-sitosterol decreased by 10.73%,the total content of 10 kinds of diterpenoids decreased 49.76% in the SmIPI1-RNAi line compared with the control group V0 line,and the difference was significant.In the SmIPI2-RNAi line,there was no significant difference in the content of campesterol and stigmasterol compared with the V0 line.The total content of the three plant sterols decreased by 6.59%.The contents of Tanshinone IIA,tanshinone IIB,15,16-dihydroxy tanshinone I,tanshinone I,1,2-dihydroxy tanshinone I,tanshinone IIA were not statistically different from those of control group V0,and the total content of 10 kinds of diterpenoids decreased by 32.35%.The results of the comprehensive study concluded that,SmIPI1 and SmIPI2 synergistically regulate the metabolism of diterpenoids on the MEP plastid pathway,and SmIPI1 plays a major role in it.SmIPI1 also modulates the isomerization of IPP and DMAPP in the cytoplasmic MVA pathway.