Expression And Immunolocalization Analysis Of Acetyl-CoA Acetyltransferase And Diphosphomevalonate Decarboxylase In Laticifers Of Euphorbia Helioscopia L.

Euphorbia helioscopia L.is a herbaceous plant with latex of Euphorbia,Euphorbiaceae,as a kind of ancient folk herbal medicines;its main medicinal ingredients are terpenoids.Terpenoids are a kind of secondary metabolites widely existing in plants.The plant terpenoids synthetizes by two main biosynthesis pathways,which are mevalonate(MVA)pathway and 2-C-methyl-D-erythritol-4-phosphate(MEP)pathway.The E.helioscopia laticifer cell is the main place for synthesizing and storing terpenoids.Based on the research of E.helioscopia latex proteomics,we have identified varieties of proteins,including acetyl-CoA acetyltransferase(AACT)and diphosphomevalonate decarboxylase(MDC),which are key enzymes of mevalonate(MVA)pathway of terpenoids synthesis.Our team has successfully cloned genes of AACT and MDC,and named EhAACT(accession number:KP995935)and EhMDC(accession number:KP995936)respectively.Furthermore,expression proteins of two genes were obtained by prokaryotic expression.In order to further understand the synthesis site,transport and accumulation of the terpenoids in laticifers,the present paper is to analyze quantitatively the expression of EhAACT and EhMDC in roots,stems and leaves of E.helioscopia by real-time fluorescent quantitative PCR(FQ-PCR),and to prepare antibodies using the obtained expression proteins,and then to analyze the immunolocalization of EhAACT and EhMDC on the levels of proteins,cells and organelle in laticifers by using of western blot and colloidal gold immunoelectron microscopy.The results above would provide theoretical foundation for the biosynthesis and regulation of terpenoids.The main results are as follows:Firstly,through the quantitative analysis on expressions of two genes EhAACT and EhMDC in E.helioscopia roots,stems and leaves by using RT-PCR technique,the results show that the expression of EhAACT has no significant difference between roots,stems and leaves,the expression level is almost the same in roots stem and leaves;the expression of EhMDC has significant difference between roots,stems and leaves(P<0.05),with expression level is higher in roots than in leaves and stems,and almost the same in leaves and stems.Secondly,antibodies were prepared using purified AACT and MDC proteins as antigens respectively,and antibody titers were detected by enzyme-linked immunosorbent assay(ELISA),the polyclonal antibody titers of anti-AACT and anti-MDC were about 1:4 00 000.Thirdly,the total protein of E.helioscopia latex was extracted,and the expressions of AACT and MDC in laticifers were analyzed with western blot.The results show that the AACT antibody identifies a stripe which molecular mass is about 43 kDa,the MDC antibody identifies a single stripe with molecular mass of about 46 kDa,which illustrate that two kinds of enzymes exactly exist in E.helioscopia laticifers,and might participate the synthesis of terpenoids in laticifers.Fourthly,the subcellular localization of AACT and MDC in E.helioscopia laticifers were observed by using colloidal gold immune-electron microscopy.Both of them were found mainly distribute in the cytoplasm,endoplasmic reticulum and vesicles originated from endoplasmic reticulum.This result provides a theoretical basis for the biosynthesis and regulation of terpenoids.