Effects Of Coronatine On Cephalotaxus Synthesis Of Cephalotaxus Mannii Suspension Cells And Its Transcriptome Analysis

To improve the yield of cephalotaxine by the callus cells of Cephalotaxus mannii and explore its synthesis pathway,the coronatine(COR)was selected to treat the C.mannii suspension cells,then compared with the cells treated by methyl jasmonate(MJ).In the course of culture,different concentrations of COR(0.5,1.0,2.0?mol/L)and MJ(50?mol/L)were added to the culture medium respectively.The effects of COR and MJ on cell growth and product synthesis were investigated by detecting the biomass,cell viability,sugar consumption of the medium,cell integrity,and G6PDH and DS enzyme activities during the culture.For make a depth analysis of the effects of coronatine on the synthesis of cephalotaxine in suspension cells of C.mannii,transcriptome analysis was used to explore the influence of coronatine and methyl jasmonate on the synthesis pathway of cephalotaxine.By analyzing the differential genes in the transcriptome,the role of elicitors in each metabolic pathway was studied,and the key enzymes in the synthesis pathway of cephalotaxine were identified.The conditions of transformation of plasmid p BI-121 mediated by Aturobacterium tumefaciens into plant suspension cells were explored,which laid a foundation for the subsequent study on the effect of related enzymes on the synthesis pathway of cephalotaxine in C.mannii cells.1.The influence of elicitor on product synthesis and cell growthWhen plant cells were treated with 1.0?mol/L coronatine,the highest yeild of cephalotaxine was obtained(6.75 mg/L),which was more than twice that of control(3.14 mg/L).Low concentrations of coronatine(0.5 and 1.0?mol/L)were beneficial to the synthesis of the products,at the mean time,it could increase the activities of G6PDH and DS within 48 h.When the concentration of coronatine was more than 2.0?mol/L,it had a strong inhibitory effect on cell growth and resulted in the decrease of G6PDH and DS enzyme activities.It indicating that the high concentration of coronatine has an inhibitory effect on cell growth.Both coronatine(1.0?mol/L)and methyl jasmonate(50?mol/L)could promote the synthesis of cephalotaxine,but the effect of coronatine was better than that of methyl jasmonate,so coronatine could better promote the synthesis of cephalotaxine in C.mannii cells than that of methyl jasmonate.2.Transcriptome analysis of inducer treatment groupTranscriptome analysis revealed pentose metabolic pathway,shikimic acid pathway,amino acid synthesis and other pathways involved in the synthesis of cephalotaxine..The elicitors affected the transcription level of some key enzymes such as DS,PAL,G6PDH,hexokinase and diterpenoid synthase.Compared with the control,the DS enzyme gene transcription was up-regulated by 3.1 times and PAL gene transcription was up-regulated by 4.53 times in the methyl jasmonate treatment group.Compared with the control group,the transcription level of diterpene synthase gene and hexokinase gene in coronatine treatment group were increased by 2.79 times and 2.86 times respectively.Compared with coronatine treatment for 24 h and 48 h,DS and PAL were increased by 2.79 times and 7.01 times at 24 h.The expression of DS,PAL,G6PDH and diterpene synthase were all highly expressed at 24 h in the comparison between the two groups treated with methyl jasmonate and coronin for 24h and 48 h.3.Agrobacterium tumefaciens-mediated transformation conditions of p BI-121plasmidThe p BI-121 plasmid was transfected into Agrobacterium tumefaciens EHA105and LBA4404,then the suspension cells of C.mannii were used as explants to transform Agrobacterium tumefaciens EHA105 and LBA4404.The transformation conditions were as follows:20 m L suspension culture cells of C.mannii were co-cultured with 10 m L Agrobacterium tumefacien LBA4404(OD₆₀₀=0.8)at 23?,50 r/min for 24 h.The co-cultures were sterilized with 100?g/m L kanamycin and 300?g/m L cephalosporin,and there was no turbidity in the culture medium.After transformation,there were about 300 bp marker gene bands in C.mannii cells,so it could be judged that the plasmid was successfully transferred.