Molecular Cloning And Characterization Of Transcription Factor Involved In Panax Notoginseng Saponins Biosynthetic Pathway

Panax notoginseng(Burk)F.H.Chen is a kind of highly valued Chinese traditional medicinal plants.Panax notoginseng saponins(PNS)are triterpenoid saponins of dammarane type.Because of the strict environment contditions and long growth time,it's difficult to develop the industry of P.notoginseng.In recent years,P.notoginseng has been used more and more widely,and the market demand was also expanded.As good natural medicine controlling cardiovascular and cerebrovascular diseases,it has important theoretical and practical value to carry out the research of PNS biosynthesis pathway.Transcription activation of gene by transcription factor is an important regulation step in the process of plant secondary metabolite synthesis.Although single key enzyme or more enzymes can be used to regulate metabolic pathway,the deficiency of the method is obvious.This is a new way to control the plant metabolic pathways by transcription factors.The AP2,WRKY,bHLH,bZIP and Zinc finger transcription factors from Catharanthus roseus,Taxus and Artemisia annua were found to participate in terpene biosynthesis.However,the research of transcription factor related to PNS biosynthesis has not been reported.In this thesis,two novel transcription factor genes,PnWRKY1 and PnbHLH1 have been isolated from P.notoginseng cDNA library through yeast one-hybrid and rapid amplification of cDNA ends(RACE).The bioinformatics analysis and functional identification are helpful for further study of the two transcription factors which may be involved in PNS biosynthesis.The bait plasmid was constructed with cis-element JERE and some positive plasmids were selected from P.notoginseng cDNA library by using yeast one-hybrid system.A cDNA-coded transcription factor gene encoding a 269-amino-acid protein with a 29.5 kD and a pI of 9.94 was named as PnWRKY1.The PnWRKY1 protein contained a single WRKY domain with one zinc finger-like motif in the C-terminal region,categorizing it into group ? of the WRKY superfamily.In addition,PnWRKY1 has a putative Leu zipper in its N-terminal end.PnWRKY1 exhibited 61%sequence identity with Panax quinquefolius WRKY10 protein.Phylogenetic analysis of the PnWRKY1 shows that it is broadly similar to AtWRKY from Arabidopsis thaliana.EMSA of PnWRKY1 analysis showed that it could bind to(T)(T)TGAC(C/T)(W-box)elements and PnWRKY1 belongs to WRKY transcription factor family.The ORF of PnbHLH1 transcription factor gene encoded a protein of 321 amino acids with a 36.1 kD and a pI of 6.97.The PnbHLH1 protein contained a single Myc-bHLH domain(140-206 aa)and belonged to secrete protein.Biological information analysis revealed that the PnbHLH1 may belong to bHLH transcription factor family.