Molecular Cloning And Functional Study Of PnERF Transcription Factor Involved In Biosynthetic Regulation Of Panax Notoginseng Saponins

Panax notoginseng(Burk.)F.H.Chen is a kind of medicinal herb in the Araliaceae family.P.notoginseng saponins(PNS)are the main medicinal activity components.Due to the special environment conditions,long growth periods and crop rotation needing,the sustainable development of P.notoginseng industry is being restricted.In recent years,the market demand for P.notoginseng is increasing year by year.The gap between supply and demand has been broadened significantly.As a special natural drug on the prevention and treatment of cardiovascular and cerebrovascular diseases,the market potential of PNS will be huge.Therefore,it is necessary to carry out basic researches relating to biosynthetic pathway of PNS.Transcription factor has the advantage of multiple regulation in plant secondary metabolism pathway.Transcription factors participating in terpenoid biosynthesis include AP2,bHLH,WRKY,Zinc finger and bZIP types.In this thesis,the degenerate primers were designed according to the ORCA family of Catharanthus roseus.By using the homologous cloning and rapid amplification of cDNA ends(RACE),an ERF transcription factor gene named PnERF was cloned from the cDNA library of P.notoginseng cell.The combinations between PnERF and promoters of key enzyme genes,the function of PnERF in PNS biosynthetic pathway were performed.Firstly,PnERF gene was got by using the homologous cloning.The gene encodes a 266-amino-acid protein with a molecular weight of 30.117 kDa and a pI of 6.09,which contains a typical AP2/EREBP domain.Multiple alignments and phylogenetic tree analysis indicated that the deduced PnERF protein had a high homology with other AP2 transcription factors.PnERF exhibited a 48%sequence identity with Catharanthus roseus ERF protein.The phylogenetic tree showed that the PnERF belongs to the ERF subfamily.The three-dimensional model demonstrated that a 71.93%similarity existed between PnERF and AtERFl coming from Arabidopsis thaliana.A 1872 bp promoter fragment of squalene epoxidase(SE)was cloned from P.notoginseng using genomic walking technique.According to the promoter sequences of dammarenediol synthase(DS)and squalene synthase(SS)in Panax ginseng,the DS and SS promoter fragments of P.notoginseng were isolated by PCR with the length were 885 and 1 276 bp.The bioinformatics analyses showed that the fragments contained the conserved promoter sequence,such as TATA-box,CAAT-box.Furthermore,it contained several cis-acting elements which related to hormones,biotic and abiotic stresses.To determine the optimal promoter sequence for gene expression,SE promoter was deleted from its 5' end to form three promoter fragments.Such fragments were fused to ? ?-glucuronidase(GUS)gene.At the same time,the plant expression vectors of the DS and SS promoter were constructed.The fused genes were transformed into Nicotiana tabacum for activity analysis of transient expression.Histochemical staining and GUS quantitative fluorometric assays demonstrated that the four different length fragments of the SE promoter had promoter activities,and the activities were increased with the deleted length of promoters.The DS and SS promoter also had promoter activities,but the activities were weaker than SE.The results of electrophoretic mobility shift assay demonstrated that PnERF protein can not only binds to GCC-box cis-elements but binds to the promoter of SE,which inferred that PnERF may participate in the regulation of PNS biosynthesis.The overexpression vector pCAMBIA 1300-PnERF was constructed and transferred into Agrobacterium tumefaciens EHA105 by CaC12 freeze-thawed method.The genetic transformation system of P.notoginseng was mediated by the A.tumefaciens.The transformed cells were screened by the hygromycin,and then thirteen cell lines which grew well were obtained.By using PCR amplification of genomic DNA,ten transformed cell lines were confirmed to be positive.Six transgenic cell lines were selected to carry out transcription analysis.The results showed that the expression level of PnERF,DS and SS genes in the six transgenic cell lines were higher than those in the non-transgenic cell line.In order to define the role and function of PnERF gene in P.notoginseng saponins biosynthetic pathway,the contents of P.notoginseng saponins in the six transgenic positive cell lines were detected.The results showed that the overexpression of PnERF in the six transgenic cell lines achieved an increase in the content of P.notoginseng saponins.In this study,six monomer saponins which were reported as major constituents were chosen to detect their contents in three transgenic cell lines.Compared to the non-transgenic cell line,except saponin Rd,the contents of the others were all much higher in the transgenic cell lines.The monomer saponins,F1 and Rg3,were not detected in non-transgenic cell lines,but existed in the transgenic cell lines.The two kinds of monomer saponins in the cells overexpressing two genes(PnERF and DS)were increased significantly.The above research demonstrated that the PnERF gene played a vital role in the biosynthesis of saponins in P.notoginseng,and the overexpression of PnERF was benefit to the accumulation of P.notoginseng saponins.