Prokaryotic Expression And Function Analysis Of DIRIGENT Gene In Eucommia Ulmoides Oliver

Eucommia ulmoides Oliver is a relict plant in the Tertiary Period,is the sole in Eucommiaceae genus,and is the single species in Eucommia family.It is mainly distributed in Sichuan,Hunan,Guizhou and other regions in China.The medicinal ingredients of Eucommia ulmoides Oliver are mainly phenylpropanoids,iridoids,lignans and other compounds.Among these lignan compounds,pinoresinol diglucoside has a bidirectional blood pressure regulating effect.Studies have shown that Dirigent protein can direct the coupling of lignin monomers to form dimers in the presence of biooxidases or oxidants,thus participate in the biosynthesis of lignin and lignan molecules.At the same time,DIR protein plays an important role in the process that responses abiotic and biotic stress factors.Therefore,the DIR protein function has important value.The study was based on the previous experiments about cloned EuDIR1 gene in laboratory.The EuDIR1 gene was studied for prokaryotic expression,heterologous and homologous genetic transformation.1.The prokaryotic expression of EuDIR1 gene and protein purificationIn this study,the prokaryotic expression vectors pET-30a-EuDIR1 and pMCSG19-EuDIR1were constructed.The recombinant vectors were transfered into E.coli BL21（DE3）to express the protein.A maximum concentration of inclusion bodies observed by induction was 0.1 mmol/L IPTG at 15℃for 6 hours.The inclusion bodies were putificated,then the target protein was collected and renatured.The His tag was identified by Western Blot and The EuDIR protein was identified by MS identification.We obtained the highly pure and soluble recombinant proteins.2.Functional analysis of EuDIR1 proteins in vitroTo investigate a potential“dirigent”activity of EuDIR1 protein we first analyzed the product spectrum generated during the oxidative coupling of E-coniferyl alcohol catalyzed by ammonium persulfate,with O₂ as the oxidizing agent.Using chiral HPLC（Chiralpak IB）the pinoresinol formed in absence of EuDIR1 protein was shown to be a racemic mixture of（+）-Pinoresinol and（-）-Pinoresinol.The spectrum of products was the same when ammonium persulfate catalyzed oxidation of E-coniferyl alcohol with O₂ as oxidizing agent was carried out in presence of EuDIR1protein.In addition,in the presence of EuDIR1 protein,the oxidative coupling of E-coniferyl alcohol resulted in the preferential formation of（+）-Pinoresinol.At the same time,The study confirmed that EuDIR1 protein has a certain inhibitory effect on Fusarium oxysporum and Valsa sordida Nits.The minimum inhibition concentrations of EuDIR1 protein against Fusarium oxysporum and Valsa sordida Nits were 912.5 and 1825μg/mL,respectively.3.EuDIR1 gene expression level in transgenic Eucommia ulmoides Oliver plantsThe plant overexpression vector pSH737-35S-EuDIR1 was constructed,which was transformed in agrobacterium LBA4404 by heat shock.Using Agrobacterium-mediated hypocotyledonary axis transformation method,five Eucommia ulmoides Oliver plants with EuDIR1 gene overexpresion were obtained via GUS staining and PCR amplification.In order to study the expression level of EuDIR1 gene in Eucommia ulmoides Oliver,the stem,leaf and root of transgenic plants,empty vector plants and wild type plants of EuDIR1 gene were determined.The results shown that the EuDIR1 gene expression levels in the stem,leaf and root of the Eucommia ulmoides Oliver plants with EuDIR1 gene overexpresion were higher than the wild type.