Effects Of ?-cyclodextrin And DNA De-methylating Agent On The Growth And Cephalotaxus Alkaloids Synthesis Of Cephalotaxus Mannii Suspension Cells

In order to increase the production of cephalotaxine,the secondary metabolite in Cephalotaxus mannii suspension cells,a noval elicitor,?-cyclodextrin(?-CD)and DNA demethylating agent 5-aza-2'-deoxycytidine(5-Aza-CdR)were selected to treat the cells.In the course of culture,different concentrations of ?-CD(5,15,25,35 mmol/L)and 5-Aza-CdR(15,25,35 ?mol/L)were added to the culture medium,respectively.The effects of ?-CD and 5-Aza-CdR on cell growth and product synthesis were investigated by measuring cell growth rate,cell viability,sugar consumption rate,cephalotaxine content and the activities of the key enzyme.For make a in-depth analysis of the relationship between DNA methylation and cell senescence and product synthesis,transcriptome studies were carried out on newly induced callus suspension cells(N),five-year-old cells(OCK)and demethylated cells(O).To analyze the differentially expressed genes and find out the genes which were related to cell senescence and product synthesis,so as to elucidate the molecular mechanism of cell senescence and synthesis of cephalotaxine in C.mannii suspension cells.The main results of this study are as follows:1.Effects of ?-CD on cell growth and product synthesisDifferent concentrations of ?-CD could significantly increase the content of cephalotaxine and promote the growth of suspension cells of C.mannii.While treated with 35 mmol/L ?-CD,the production was the highest(2.26 mg/L),which was 4.18 times than that of the control(0.54 mg/L).The suspended cell biomass was 14.79 g/L,1.5 times than that of the control(9.79 mg/L).At the same time,P-CD can effectively enhance the activity of glucose-6-phosphate(G6PDH),which is the key enzyme of the Hexose monophosphate pathway(HMP pathway),and the rate-limiting enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase(DS)of Shikimic acid pathway.2.Effect of 5-Aza-CdR on cell growth and product synthesisThe cell growth and product synthesis in suspension cultures cells were promoted when treated with different concentrations of demethylation reagent 5-Aza-CdR.Addition of 15 ?mol/L 5-Aza-CdR increased the product content by 29.67%and cell dry weight by 13.75%.At the same time,5-Aza-CdR can effectively improve the activities of pyruvate kinase(PK),G6PDH,DS and provide more "Carbon sources" for product synthesis.It was found that 5-Aza-CdR could decrease the methylation level of cells by measuring the degree of methylation in the cell culture cycle.During the culture process,the methylation trend of cells decreased first and then increased.Demethylation mainly occurred in the early growth stage(0-1 5 d)when treated with 5-Aza-CdR.3.DNA methylation was associated with cellular senescence and product synthesisBy transcriptome analysis,it was found that DNA demethylation could reduce the expression of protein phosphata(PP2C),the auxin negative regulator auxin/indole-3-acetic acid(Aux/IAA),the small auxin up RNA(SAUR)and increase the expression of auxin response factor(ARF)in cells,thereby improving the ability of cells to resist stress,slowing down cell aging and promoting cell growth.At the same time,DNA demethylation could increase the expression of genes which are related to fructokinase and aldolase in glycolytic pathway,that provided more ATP and phosphoenolpyruvate to promote cell growth and product synthesis.