Molecular Cloning And Functional Study Of Transcription Factors TcMYC And TcWRKY1That Regulate The Taxol Biosynthesis

Plant cell cultures is one of the most promising method to solve theshortage of rare secondary metabolites, however, the yield of secondarymetabolites in plant cell cultures is low, which limited its commercialapplication. One of effective approaches is metabolic engineering of plantcell in an attempt to obtain a higher secondary metabolites content.Transcriptional factors can activate multiple expression of genes encodingsynthases in biosynthetic pathway of certain secondary metabolite, improvingthe content of this secondary metabolite. Taxol (paclitaxel) is an efficientantitumor agent originated from yew trees and has been widely used in thetreatment of breast, ovary and other cancers. Taxus suspension cells culture issuitable for the commercial sources of taxol due to its renewable andsustainable production system. However, the yield of taxol is low in culturedTaxus cells, which limited the application of cell culture. In this research, theMeJA and SA-induced Taxol synthesis key enzyme genes were chosed as model, and the promoter of gene response to MeJA and SA was cloned,through5’ deletion analysis identified the MeJA and SA-responsed elements,and then isolating the binding protein from the T.chinensis cells by the yeastone-hybrid system using MeJA and SA-responsed elements as baitrespectively, Constitutive expression and RNAi of the binding protein in T.chinensis cells were used to study the function of these genes. Constructedthe alcohol inducible transcription factor expression vector, which was thenintroduced into T. chinensis cells for achieved the transgenic Taxus cells withcontrollable expression of transcription factors regulating taxol biosynthesis.The innovative results of this study were as follows:(1) The expression patterns of the key taxol biosythesis genes responsed toMeJA and SA were obtained by quantitative PCR. It is found that most of thetaxol biosynthesis genes were induced by MeJA, especially for thepreliminary steps such as ts、t5αh、tat, the late steps gene t13αh，tbt，dbat，dbtnbt，bapt can be significantly induced by SA. (2) The promoter sequence of taxol biosythesis gene ts and dbat werecloned from T. chinensis.Using PLACE and PlantCARE database analysisfound that the promoter of ts gene contained typical JA responsive elements,including the G-box region, TGACG and GCC-box, while the promoter ofdbat contains a typical SA response elements such as W-box.The functioncharacteristics of ts, dbat promoter was analyzed, GUS analysis showed thatthe ts, dbat promoter can drive GUS gene expression in tobacco and T.chinensis cells, GUS quantitative analysis showed that the (-940/-240)element of dbat promoter was significantly induced by SA, the (-239/-131)element of ts promoter were significantly induced by JA.(3) A WRKY family transcription factor named TcWRKY1was isolatedusing the SA response element(-940/-240) as bait, and a bHLB familytranscription factors named TcMYC was isolated using the JA responseelement(-239/-131) as bait, Subcellular localization experiments show thatTcWRKY1and TcMYC were located in the nucleus, EMSA experiments showed that TcWRKY1can bind to the W-box of SA response element(-940/-240) in vitro. The constitutive expression and RNAi experimentdemonstrated that TcWRKY1can regulated the expression of dbat gene, andthe overexpression of TcMYC in T. chinensis cells showed that the TcMYCcan activation the ts gene expression.(4) Construction the alcohol-induced transcription factor TcWRKY1expression vector using the transcription factor TcWRKY1as the target genes,which was introduced into the T. chinese cells to study the the expressionchanges of key enzyme and content of taxanes. After alcohol induce, theexpression of ts，t5αh，dbbt，dbat，dbtnbt was enhanced, and taxol yield wasabout2.7times that of the uninduced cells.