Study On The Effect Of Light Stress On Taxanes Conent Of Taxus Mairei (Lemee Et Levl.) S.Y.Hu Ex Liu And Its Molecular Mechanism

Objective To establish a HPLC-DAD method for simultaneous determination of 10deacetylbaccatin Ⅲ and taxol content of Taxus mairei(Lemee et Levl.)S.Y.Hu ex Liu.To establish a HPLC-ELSD method for determination of yunnanxane content of Taxus mairei(Lemee et Levl.)S.Y.Hu ex Liu.Determination of Taxus taxanes biosynthesis key enzyme gene expression method to establish fluorescence quantitative PCR method.Determination of 5 months under different shading conditions of Taxus chinensis(2015.6.28-2015.11.28)changes of taxane content during growth.The accumulation of taxanes under different shading conditions,provide a theoretical basis for determining the optimum shading conditions of Taxus chinensis.Determination under different shading conditions of Taxus chinensis var.mairei TH5α,7 β,140H,Bapt etc.four taxane synthesis key enzyme expression,correlation of different shading conditions taxanes and synthesis key enzyme gene expression,clarify molecular mechanisms of different shading conditions affect the taxanes biosynthesis,provide a theoretical basis for the semi synthetic technology,promote the sustainable development of Taxus chinensis var.mairei.Methods ①Established of Taxus chinensis var.mairei taxane 10 de acetyl baccatin Ⅲ,taxol two components and method to determine the content of taxanes by HPLC-DAD method.②Using HPLC-ELDS method to establish a method for determination of taxanes in Taxus yunnanxane,determination of taxane content.③Establishment of fluorescent quantitative PCR was used to detect the four kinds of taxane biosynthesis key enzyme[TH5α,7β,140H,Bapt)enzyme gene expression.④Are respectively arranged 0%,55%,75%,90%of four different shading conditions,using the five point method,collected from 5 years old plants of Taxus chinensis var.mairei in parts of foliage samples,through the determination of 5 months growth process in 6 batches(monthly batch samples taxanes content.Under conditions of different shading on the South yew leaves taxane accumulation change rules.⑤By fluorescence quantitative PCR method for determination of growth process of Taxus chinensis var.mairei taxane biosynthesis key enzyme gene expression variation under different shading conditions for 5 months,combined with the taxane content changes,to explore under different shading conditions of Taxus chinensis var.mairei taxane content a molecular mechanismResults ①Two kinds of the taxanes determination method of optimal chromatographic conditions for:inertsil ODS-SP(250×4.6mm,5μm);The mobile phase was methanol-water(0-16min,44%;16-20min,44%-62%;20-25min,62-67%;25-30 min,67%-70%;30-35 min,70%-62%;35-40min,62%);The flow rate was 0.5mL·min-1;theUV detection wavelength was 227 nm,and the column temperatu the re was 30℃.The Sample quantity was 10μL.In the chromatographic conditions,the target component separation between each adjacent peak degrees were greater than 1.5,trailing factor between 0.95-1.05;Theoretical plate number greater than 30000,It can satisfies the requirement of quantitative.② Yunnanxane of the taxanes determination method of optimal chromatographic conditions for:C18 column inertsil(250×4.6mm,5μm);The mobile phase was methanol-water(0-8min,20%-100%;8-18min,100%);The flow rate was 1mL·min-1;the column temperatu the re was 25℃.The Sample quantity was 20μL.ELSD gasification room temperature:45℃,nitrogen pressure:0.4MPa.In the chromatographic conditions,the target component separation between each adjacent peak degrees were greater than 1.5,trailing factor between 0.95-1.05;Theoretical plate number greater than 30000,It can satisfies the requirement of quantitative.③T According to different shading conditions under of Taxus chinensis var.mairei 10-DAB III,paclitaxel,yunnanxane three components of content detection,through the foliage in content with time change analysis and found to appear a certain correlation.In the 51.6%group,the content of 10-DAB Ⅲ and paclitaxel were increased,and the content of yunnanxane was significantly decreased.According to the experimental results,the optimum conditions for the determination of Taxus chinensis shade 50%.④Through the detection of the four groups under shade conditions of Taxus chinensis var.mairei TH5 α、7β、140H、BAPT and four taxane biosynthesis key enzyme gene expression and combined with the first half of the 4 groups under shading condition 3 kinds of taxane content dynamic change research results were analyzed,external stimuli greatly affected the key enzyme gene expression,thereby regulating taxane synthesis.This experiment found that 51.6%shading can increase the degree of TH5 α,7 β gene expression,and 69%shading treatment on BAPT has great influence.Conclusion The yunnanxane component determination method of the South China is established.The method is simple,rapid,stable and controllable,has strong adaptability,good reproducibility and accurate measurement results,and can meet the requirements of content detection;Under different shading conditions,Taxus chinensis var.mairei taxane content accumulation change present certain regularity,showing certain correlation,and from the point of view of gene to explore the mechanism of its content is not consistent.This study has a certain guiding significance for the planting and harvesting of the South China.