Subcellular Localization And Functional Analysis Of EuTPT5 And EuREF1 Gene Expression Products In Eucommia Ulmoides

Eucommia ulmoides gum（EUG）is a kind of trans-polyisoprene（TPI）biosynthesized by Eucommia ulmoides,which is widely distributed in roots,stems,leaves,bark and pericarp of E.ulmoides.The chemical composition of EUG is the same as that of natural rubber（NR）,and they are cis-trans isomers,that is,NR is cis-1,4-polyisoprene（CPI）,while EUG is trans-1,4-polyisoprene.During the biosynthesis of EUG,the polymeric precursor isopentenyl pyrophosphate IPP is generated by the mevalonate（MVA）pathway;under the action of isopentenyl pyrophosphate isomerase IDI,part of IPP is isomerized to DMAPP;IPP and DMAPP IPP and DMAPP are polymerized and condensed to form a series of oligomeric trans polyisoprene.Pentenyl transferase can catalyze the sequential condensation of IPP with FPP(C₁₅)as the main substrate.According to the stereochemistry of the double bonds formed during the condensation of IPP,these Pentenyl transferases can be divided into cis-prenyl transferases（CPTs）and trans-prenyl transferases（TPTs）.On the surface of natural rubber particles,CPT forms membrane-binding complexes with other functional proteins to produce ultra-high molecular weight CPI.However,the molecular mechanism by which TPT is involved in the synthesis of TPI remains unclear.In order to understand the molecular mechanism of TPI biopolymerization,47 key EUG biosynthesis genes were identified in E.ulmoides genome,and their expression changes were analyzed.The transcript level of EuTPT5 gradually increased during the fruit development stage,which completely corresponded to the accumulation of TPI in the pericarp.However,as the fruit entered the ripening process,although the expression level of EuTPT5 decreased,the TPI content in the pericarp changed little after reaching the peak,which confirmed that there was a correlation between the production of TPI and EuTPT5.In addition,through the comparative analysis of EuREF1（rubber elongation factor,REF）gene expression and gum content of E.ulmoides in different periods,it was found that EuREF1 expression was positively correlated with the growth of EUG.It is speculated that EuREF1 gene expression products may play an important role in the extension of rubber chain.The purpose of this study is to explore the subcellular localization of EuTPT5 and EuREF1 gene expression products through Agrobacterium-mediated methods,observe and analyze their respective subcellular localization,and to observe and analyze their respective subcellular localization to determine whether they are spatially close to each other and whether they have the basis of interaction.At the same time,the genetic transformation and regeneration system of N.benthamiana was established,and the EuTPT5 and EuREF1 genes were transferred into N.benthamiana,and the gene expression and TPI production were analyzed and compared,so as to determine whether EuTPT5 and EuREF1 jointly promoted the biopolymerization of TPI,which provides a theoretical basis for further revealing the mechanism of TPI biosynthesis.The research results of this paper are as follows:1.Extract the total RNA of E.ulmoides pods,reverse transcription and PCR amplification to obtain the CDS fragments of EuTPT5 and EuREF1 genes.The sequencing results are consistent with the sequence results published by NCBI.2.Online prediction of the subcellular localization of EuTPT5 and EuREF1 gene expression products.Construct 35S:EuTPT5:EGFP,35S:EuREF1:EGFP transient expression vector.The transformation solution was injected into tobacco leaves by injection method,and after dark culture,the epidermal cells under the monolayer were torn and sliced,and the subcellular localization of EuTPT5 and EuREF1 proteins was observed under a laser confocal microscope.The results showed that the expression products of EuTPT5 and EuREF1 were both located in the cytoplasm.3.The genetic transformation vectors 35S:EuTPT5:NOS,35S:EuREF1:NOS and the bivalent expression vector 35S:EuTPT5:EuREF1:NOS were constructed,and the target gene was transferred into N.benthamiana by Agrobacterium-mediated method to obtain transgenic positive plants.The DNA of the transgenic positive strains was extracted for PCR verification,and 4 double-transformed（EuTPT5,EuREF1）positive strains,4 EuTPT5positive strains,and 7 EuREF1 positive strains were identified.The relative expression levels of EuTPT5 and EuREF1 genes were analyzed,and the results showed that compared with wild-type plants,the relative expression levels of EuTPT5 and EuREF1 genes in transgenic positive plants were significantly increased.4.The TPI components of transgenic plants were extracted by 2 m L extraction method,and the molecular weight distribution of the extracted products was analyzed by gel permeation chromatography（GPC）.The TPI of the transgenic plants was extracted by Soxhlet method,and its content was determined.Experiments show that EuTPT5 has TPI synthesis activity,but EuREF1 does not have trans-polyisoprene synthesis function.At the same time,EuTPT5 can increase the molecular weight of TPI components,while EuREF1does not promote the relative molecular weight of TPI components.