Molecular Cloning And Characterization Of AP2-Type Transcription Factors Involved In Isoprenoid Biosynthetic Pathway Of Taxus Cuspidata

Isoprenoid biosynthetic pathway plays an important role in plant terpenoid natural metabolism pathways and terpenoids' synthesis.Most of the products (Artemisinin,Taxol,Ginkgolide) in isoprenoid biosynthetic pathway have been broadly applied in medical field and clinical therapy for human beings.Therefore, studying the isoprenoid biosynthetic pathway in Taxus cuspidata is important for further understanding the regulation and biosynthesis of taxol,the important anti-cancer chemical produced in Taxus trees.Taxus is an ancient and precious species.Taxol(paclitaxel) is one of natural diterpenoid alkaloids firstly isolated from the bark of the yew(Taxus brevifolia). Taxol has been well established and approved by FDA as a very important effective chemotherapeutic agent against a wide range of tumors since 1992.However,the supply of Taxol has been limited since the discovery of this natural product,and with increasing applications in chemotherapy,the availability and cost of the drug will remain an important issue.Transcription factors(TFs),found to be regulators of metabolic flux,have become a powerful tool for the manipulation of complex metabolic engineering in plants.In this thesis,two novel AP2 TF genes,TcAP2 and TcDREB,which may be involved in taxol biosynthetic pathway,has been isolated from Taxus cuspidata for the first time.Probing into the induction effects of methyl jasmonate(MeJA) and salicylic acid (SA) to taxol content,suspension cells of T.cuspidate were treated with exogenous MeJA and SA before construction of yeast one-hybrid library.Based on spatial distribution frequency of C-13 phenylpropanoid side chain-CoA acyltransferase (BAPT),we explored the optimal induction time of MeJA and SA to the suspension cells for the construction of cDNA library.The bait plasmid was constructed with cis-element JERE and 72 positive plasmids were selected from T.cuspidata cDNA library by using yeast one-hybrid system.A cDNA-coded TF encoding 268 amino acids was obtained.It had a conserved AP2/EREBP domain in which the N-terminal had a 4-amino acid nuclear localization signal(NLS) and C-terminal had a transcriptional activation domain, named as TcAP2(GenBank accession number EU549860).Multiple alignments and phylogenetic tree analysis indicated that TcAP2 was a member of AP2/EREBP transcription factor gene family and the deduced TcAP2 protein had the highest homology with At1g21910 from Arabidopsis thaliana and PpAP2 from Physcomitrella patens,which possibly reflected the similar functions among them. The binding site V14 inβ-sheet of YRG element and A37 in or-helix of RAYD element was conserved in AP2/EREBP domain which reflected its binding ability to DRE cis-element and GCC Box.Via SMART Rapid Amplification of 5' and 3'-cDNA Ends(RACE),another novel AP2 transcription factor gene,TcDREB(GenBank accession number EU549861),was cloned from T.cuspidata.The full-length cDNA of TcDREB contained a 1233-bp ORF,encoding 410 amino acids with no N-terminal propeptide. Predicted tertiary structure showed it had the conserved AP2 domain like TcAP2. Multiple alignments showed TcDREB was also a member of the DREB subfamily. TcDREB also possessed threeβ-sheets and oneα-helix as well as V14 and A37 DNA binding sites.Subcellular location analysis of TcAP2 and TcDREB demonstrated that they were nuclear-localized proteins.Semi-quantitative RT-PCR revealed that TcAP2 and TcDREB constitutively expressed in all test tissues,including stems,roots,and leaves, with high expression in new stems.EMSA of TcAP2 and TcDREB analysis indicated that both of them could bind to DRE element.Besides,TcDREB could bind to the promoters of 10β,13α,5αand TS as well as GCC Box of TS promoter in taxol biosynthesis pathway but not to MGCC Box.Determination of binding specificity was testified by AFM force measurement. The mean value of single molecular interaction force of TcDREB-DRE was determined as(76.3±2.3) pN.The binding probability was(19.3±5.2).The probabilities of TcDREB and other four promoters were ranged from 15%to 20%, suggesting the regulation of TcDREB in taxol biosynthesis pathway of T.cuspidata.The expression of TcAP2 and TcDREB could be induced by MeJA,low temperature and salt,ABA and salt,suggesting their involvement in isoprenoid biosynthetic pathway and ABA-dependent signal transduction.In order to analyze the regulation of the two transcription factors in isoprenoid biosynthetic pathway,the over-expression vector containing TcAP2 or TcDREB cDNAs driven by the CaMV 35S promoters and vectors carrying TS/5α/10β/13α-promoter-GUSA were constructed and single or co-transformed into A. thaliana.Transgenic plants transformed with TcDREB over-expression vector were generated,which will be used to test the effects of stress on electrolyte leakage rate in the transgenic plants in further study.In summary,the cloning and analysis of TcDREB and promoters of 10β,13α,5αand TS provide the basis for further studying the detailed role of TcDREB in regulation of taxol biosynthetic pathway of T.cuspidata.