Regulation Mechanism Of DNA Methylation On Taxol Biosynthesis In Taxus Chinensis Cells

The instability production of the secondary metabolites in the process of plant cellculture restricted its industrialization process. Previous studies have shown that the Taxuspaclitaxel synthesis in cell culture and DNA methylation level was significantly negativelycorrelated, the regulatory mechanism was not very clear. In order to reveal the regulatorymechanism, the proteomic differences between Taxus chinensis cells untreated with5-Aza-CdR and treated with5-Aza-CdR were analysised. The main results were asfollows:1)Established a special HPLC method to determine the methylation level of Taxuschinensis cells. The critical methodological factors effect HPLC analysis of plant DNAmethylation were examined. The optimal condition was as follows: DNA was hydrolyzedwith DNA degradase at37℃for3hours. The mobile phase was solvent A （50mmol/Lpotassium dihydrogen phosphate: tirethylamine=100:0.2）, and solvent B （methanol）.The gradient was set as10%B. The wavelength was285nm. HPLC results under the abovementioned conditions had good stability and repeatability.2)Optimization of Two-dimensional Electrophoresis Technology System for Taxuschinensis cells. Three different protein extraction/solubilization methods a modifiedtrichloroacetic acid （TCA）/acetone, phenol, trichloroacetic acid （TCA）/acetone combinephenol were compared. TCA methods showed higher protein resolution and spot intensityof all proteins compared with the other two methods. In addition, the results showed thatusing TCA/acetone protein extraction method, pH4-7IPG strip and improved IEFprocedure about2000protein spots were detected and the protein plots were dispersed andclear. These results suggest that the TCA methods are efficient and reliable methods fortwo-dimensional electrophoresis （2-DE） separation of Taxus chinensis cells proteins.3)Totally121protein spots changed more than1.5times were found by2-DEtechnology. Using Matrix-Assisted Laser Desorption/Ionization Time of Flight MassSpectrometry （MALDI-TOF-MS） method,29protein spots and33predicted protein weresuccessfully identified, and the29protein were involved in the regulation of Defense/Desease, Carbohydrate Metabolism, Energy Metabolism, Lipid Metabolism,Secondary Metabolites, Cell Growth and Death, Transport and Catabolism and Other.4)Cloning and expression analysis of differentially expressed protein and taxolsynthesis related enzyme gene by fluorescence quantitative RT-PCR technology. Theresults showed that gene were cloned according to the sequences obtained fromtranscriptome of Taxus chinensis cells. Further research by quantitative real-time RT-PCRshowed that caffeoyl-CoA-O-methyltransferase（F14） gene gene have same expressiontrends with protein, AT5g62700/MRG21₁2（A3）gene, unknown（C3） gene, tyrosine3-monooxygenase/tryptophan5-monooxygenase activation protein（C4） gene,Full=S-adenosylmethionine synthase2; AdoMet synthase （D14） gene,, Hsp90（E3） geneand actin2（M1） gene however, showed poor correlations between transcript and proteinlevels at particular time points. This inferred that most of the genes were also regulated atthe transcript Level.5-Aza-CdR having a greater impact on the expression ofmethyltransferase MET5gene, but a minor effect on the methyltransferase MET1andMET4, this inferred that5-Aza-CdR have different effects on different methyltransferasegenes.5-Aza-CdR also could improve the expression level of taxol biosynthesis gene TS、DBAT、BAPT.5) The regulation mechanism of methylation on taxol biosynthesis werepredicted,according to the results of transcript and protein.5-Aza-CdR was able to reducemethylation and able to activate the expression of proteins that were involved in Energymetabolism, Pentose phosphate pathway, Glycolytic pathway, Fatty acid metabolism, andother biological functions. Those metabolism could provide NADPH, energy, Acetyl-CoA,Glyceraldehyde3-phosphate to the process of taxol biosynthesis.5-Aza-CdR was able toactivate the expression of taxol biosynthesis genes（TS、DBAT、BAPT）. In addition taxolproduction in suspension cultures could induce cell apoptosis in suspension cultures ofTaxus chinensis cells. This leads to decreased activation of5-Aza-CdR in turn.So activatedgenes were remethylated again and silence expression in the later stages of processing..