Cloning And Genetic Transformation Of EuDIR3, A Gene Coding Dirigent Protein From Eucommia Ulmoides Oliver

Eucommia ulmoides Oliver（EU）belongs to the family of Eucommiaceae,which is a tertiary relict plant.Its leaves and bark can be used in traditional Chinese herbal medicine.EU is rich in different active chemical composition and has many medicinal functions.In addition,EU is an important propolis plants.Dirigent protein（DIR）not only plays an important role in the synthesis of plant secondary metabolites in plants,but also participates in the anti-adversity of plants.The research on the Dirigent gene of EU can provide theoretical reference for its development and utilization.The EuDIR3 were cloned based on the EU transcritome and genome database.Then,the genetic transformation and expression analysis of EuDIR3 were carried out.The following results were obtained:1.EuDIR3 cloning and sequence analysisUsing the tender leaf of EU as the experimental material,the RNA was extracted and reverse transcribed into cDNA,which was used as a template for PCR amplification,and finally the EU dirigent protein coding gene（EuDIR3）was cloned.The full sequence of this gene was 351 bp in length,which encodes 116 amino acids and contains no introns.Homology analysis shown that EuDIR3 had high homology with Coffea eugenioides,Ananas comosus and Ricinus communis.Phylogenetic tree analysis shown that the EuDIR3 had close relationship with Daucus carota and Ananas comosus,but had a distant relationship with Prunus mume and Prunus redoensis.Bioinformatics analysis shown that the EuDIR3 had a molecular weight of 11.84 kD and a theoretical isoelectric point of 4.66,which is an unstable hydrophobic protein;This protein is distributed in the cytoplasm,mitochondria and nucleus,with the most distribution in the cytoplasm（56.5%）;The secondary structure of this protein contains 6 beta sheets but no alpha helix,where the beta sheets were connected by 7 random coils.In addition,total of 22 phosphorylation sites was detected in this protein which were throughout the polypeptide chain,but no signal peptide and transmembrane structure.2.Spatial expression pattern of EuDIR3In order to analyze the spatial expression characteristics of the EuDIR3,Elongation factor1 alpha（EF1α）was selected as the housekeeping gene and Real-time PCR was used to analyze the relative expression of EuDIR3 in the EU bark,pistillate flower and leaves at flowering stage and root,stem and leaves at seedling stage.The results shown that:At flowering stage,the expression level of EuDIR3 in the bark was significantly higher than that in pistillate flower and leaves.In the seedling stage,EuDIR3 had the highest expression in leaves,followed by root and stem.It is speculated that EuDIR3 had a greater influence on bark at the flowering stage,but had a greater influence on leaves at the seeding stage.3.Construction of prokaryotic expression vector and expression of EuDIR3The recombinant plasmid of pET-28a-NAD1 was used as the initial vector to construct prokaryotic expression vector of pET-28a-EuDIR3 by one-step cloning method.The recombinant plasmid of pET-28a-EuDIR3 was transformed into E.coli BL21（DE3）strain.After induced with 1mM isopropylthio-β-D-galactoside（IPTG）for 12 h at 21°C,a large amount of the target protein is obtained,and the protein is mainly composed of inclusion bodies in the precipitate.The recombinant protein was identified by 15%sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）,and a specific protein band was detected at a relative molecular mass of about 15 kD,which is the recombinant protein EuDIR3.4.Construction of EuDIR3 plant expression vector and genetic transformation of Eucommia ulmoidesThe recombinant plasmid of pGM626-Act1-EuABP2 was used to construct the plant expression vector of pGM626-Act1-EuDIR3 by one-step cloning method.The gene EuDIR3were controlled by Act1 promoter and a Bar::GUS gene were contained as the selectable marker.In order to increase the yield and rooting rate of adventitious buds of EU,B₅,MS and WPM media were used to culture genetically transformed explants.Using EU hypocotyls as explants,Agrobacteriumtumefaciens-mediatedpGM626-Act1-EuDIR3and pSH737-35S-EuDIR1 were used to genetically transform EU,and each explanted explants were inoculated on three different media.The growth of explants on each medium was recorded,and the number of positive buds was obtained by GUS staining and PCR detected.Results indicated that:（1）The average germination rates of the transforming plasmids on B₅,MS and WPM media are 14.79±6.20%,7.68±2.37%and 4.05±0.64%,respectively.Compared with MS and WPM media,callus on B₅ medium grew vigorously with bright green color and tight crisp texture,whose germination rate is significantly higher than the other two media.（2）The rooting rate of WPM,B₅ and MS medium were 53.85%and 3.57%,respectively.Obviously,the rooting rate of WPM is significantly higher than the other two media,Therefore,B₅ medium is more suitable for inducing adventitious buds during the genetic transformation of EU,and WPM medium is beneficial to rootage of adventitious buds.