The Formation Rule And Regulation On The Hydroxyl And Acyl Products Of Taxane In Taxus Chinensis

Taxol is a kind of natural broad-spectrum anti-cancer drug. The technology of plant cell culture is condidered to be the most promising approach which industrialize the production of taxol at present, but there also has some problems like the yield of taxol and its pharmaceutical precursor is very low while the by-product is high. From the view of secondary cell metabolism, from geranylgeranyl pyrophosphate(GGPP) of terpenoids’ common precursor to taxadiene, then to taxol, the plant cell has experienced some complex biosynthetic reactions with a series of hydroxylations and subsequent acylations as the main functional groups. Taxol and its pharmaceutical precursor are one particular branch of the complex metabolite fluxs. The existing research shows that formation of taxane’s functional group is controlled by the specific genes, and affected by the reaction kinetics. The gene family of hydroxylation and acylation show the networked control characteristics, but the distribution of these networks and controlled factors are still unclear, so we can not regulate the synthesis of taxol effectively. This research analysed the related metabolic network in Taxus chinensis cell using method of metabonomics combined whih the deep analysis of the transcriptome data, aimed to illustrate the formation rule of taxane’s functional group in Taxus chinensis cell, and found the key nodes or regulation in the network which could effectively control the synthesis of taxol and its analogues, provided theoretical and experimental basis for efficient directional synthesis of taxol using technique of plant cell culture finally. The main work is as follows:(1) Established an metabolic profile analysis method of taxane compound. Extracted taxanes step by step using methylene chloride, methanol and water, which can keep taxanes of different polarity in the plant cell more comprehensive. Established adetection analysis method using reverse phase phenyl column (Zorbax SB-Pheny1,4.6mm*250mm,5μm) as the separation column, polar solvent of acetonitrile and water as the mobile phase, UV spectrum and mass spectrometry as the detector, which could analyse intracellular micro taxanes semi-quantitatively and simultaneously. Established a taxane LC-MS database of Taxus chinensis cell using our method. (2) Established a systematical metabolic network research method using metabonomics combined with gene transcriptome analysis. Analyzed the primary metabolites and key intermediates metabolites of terpenoids synthesis in Taxus chinensis cell using gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) technique and metabonomics, gained the metabolic data information of about209metabolites. Got the secondary metabolites data information of taxane within the cell using liquid chromatography-electrospray ionization-mass spectrometry(LC-ESI-MS)as the taxane metabolic profile analysis method. Selected a set of related hydroxy and acyl gene in taxol biosynthesis using our transcriptome data of Taxus chinensis cell which had been completed, which were analysised synchronized using quantitative real-time polymerase chain reaction(QRT-PCR) technology to gain the metabolic data of multiple related genes. Used a strong inducer of taxane biosynthesis, called methyl jasmonate (MJ) and dissolved oxygen as tools of metabolic disturbances. We got multivariate analysis based on metabolic and gene transcription data, mapped the metabolic network diagram between the primary metabolite and secondary metabolite of taxane in Taxus chinensis cell, got the metabolic rule and regulation method of different taxanes in Taxus chinensis cell under dissolved oxygen treatment.(3) Analyzed the hydroxyl and acyl of taxane formation in Taxus chinensis cell. Found that the yield of taxol group(TAX) rised more than that of multiple acetyl taxane (MAT)after MJ induction. While also found that the gene transcription level of the selected20kinds of taxol synthesis related hydroxylation and acylation appeared similar to the products. Our research also found that the total hydroxyl gene expression was far higher than the total acylation gene expression with the incremental rulethat increment of TAX was higher than that of MAT. Principal component analysis (PCA) results showed that the increasement of hydroxyl gene expressioncould help metabolism flow to TAX, and the two unknown gene of OHX1and ACX3were likely to play an important role in rising TAX and reducing MAT.(4) Analyzed the influence from the dissolved oxygen to the metabolic flow in Taxus chinensis cell and its regulation. Gained the metabolic information of209intracellular primary metabolism and intermediate using metabolomic analysis method, comprehensively contacted the data between the intracellular metabolites of taxane and7 related genes in QRT-PCR. Found that the expression of hydroxyl gene under LDO was higher than that two groups and TAX increased significantly under LDO. Further analysis found that under the condition of low dissolved oxygen, glycolytic pathway and pyruvate anaerobic pathway enhanced, Tricarboxylic acid cycle weakened, the biosynthesis of mevalonic acid pathway to synthetize terpenoids enhancee at the same time, the threonine metabolic pathway which from pyruvic acid to synthesis of cytochrome C also enhanced. Combined the change rules of metaboliem product and the related genes, we speculated that the enhancement of terpenoids synthesis pathway and hydroxyl gene expression level under low dissolved oxygen might be prime reason which lead to the change of taxane.