The Effect Of Temperature And Its Interaction With MeJA Treatment On The Synthesis Of Triterpene In Betula Platyphylla Suk.

Suspension-cultured cells of birch were experimental materials in this paper.We investigated effects of the temperature stress and their interactions with MeJA treatment on the growth of birch cell,accumulation of triterpene,defense system startup and the regulation of the key enzyme genes expression in birch triterpenoid biosynthesis pathway;At the same time,in order to obtain more stable birch triterpene material source,we used callus of birch to induce cluster seedlings,and explored triterpene substances accumulation characteristics of cluster seedlings and the response to the temperature stress.This study to lay the foundation of the environmental regulation mechanism of efficient synthesis of birch cell triterpenoid and the bioreactor large-scale production practical technology,the results were as follows:1.Different temperature stress promoted the accumulation of the total triterpenoid,oleanolic acid in birch suspension cells,and the total triterpenoid has the highest accumulation of 56.93 mg/g after 3h stressed in high temperature of 50?,then cultivated for 48 h,about 63.59%higher than control.Cultivating 24 h and 72 h after low temperature stressed in 4? for 3 h,contents in are higher as well.The total triterpenoid content are 45.60 mg/g and 52,79 mg/g,43.70%and 26.79%higher than control respectively.50? treatment for 3 h and cultivating 24 h promoted the accumulation of oleanolic acid significantly.Its content is 0.896 mg/g,92.20%higher than control.4? Low temperature stressed for 1h and 3h after early renewal cultivating(0-6h)significantly promoted the oleanolic acid accumulation.The effect in anaphase is not good.2.High temperature with Me JA treatment have different degrees of promoting function on the accumulation of birch triterpene.Cells were incubated in 50 ? for 3h after 1d the MeJA added,then recovery cultivating 36 h-72 h,the accumulation of total triterpenoid are higher than control,and the results are significant difference with control and other treaments.The total triterpenoid accumulation of 36 h reaches a maximum,about 85.62 mg/g.And it increases by 106.11%,66.97%and 84.72%respectively than that of control,MeJA and MeJA + 50 ? 3 h.Which illustrates that using appropriate high temperature stress(50?3h)after MeJA inducing for a certain time can give full play to the role of the two kinds of abiotic stress factors,and more conducive to improve total the triterpenoid content.3.On the 7th-day after birch cells were cultured,we use high temperature 50? and low temperature 4? treat those cells for 1h and 3h respectively,then renewal cultivating again,to detected PAL,SOD,CAT and APX activity change of the cells.Results showes that the high and low temperature stress stimulated response of defense system in birch cells to different extent,and the active oxygen scavenging enzymes change significantly.After high temperature with MeJA processing,every treatment improved the activity of PAL,SOD,but the timing points of reaching the peak are different.After high temperature with MeJA processing,the activity of APX were lower than control's.Which indicates that temperature stress and temperature interacts with MeJA stimulated the active oxygen scavenging enzyme system startup in suspension cells of birch,the interactions of PAL,SOD,CAT and APX these four enzymes can protect cells and stimulate the accumulation of secondary products.4.Real-time quantitative PCR was used to detect the changes of the expression of key genes in the birch terpene synthesis pathway,after temperature stress and their interactions with MeJA treatment.Results show that the key genes in birch triterpene synthesis pathway and stress tolerance related genes have different response under temperature stress and their interactions with MeJA inducement.Between the high temperature and low temperature stress individual treatment,the effects of genes expression treated by the low temperature stress is more significant.While MeJA and 50? high temperature stress treated at the same time,has the most significant effect on the expression of BPY,BPW,SS,HSP70 and ACC genes.These genes were up-regulated to different degrees,which may be the main reasons of the accumulation of triterpene.5.Established the technology regulating the synthesis of triterpene and flavonoids through the cultivation of suspended cluster seedlings of birch.The culture technology of cluster seedling of Betula Platyphylla:In this study,solid medium with NT + 2mg/L 6-BA has the best effect on inducing the cluster seedlings of Betula Platyphylla,and a culture system,by which the cluster seedlings could grow stable and has a high ability of producing the triterpene,has been established.Temperature regulating the sytnhesis of tritepene and flavoniods technology:The content of the total triterpene,oleanolic acid and the total flavoniods in the cluster seedlings were 76.41 mg/g(10d),0.53mg/g(15d)and 0.87mg/g(20d)respectively..The correlation analysis results of stress-tolerant enzymes activities and the secondary product accumulation in the cluster seedlings shows,the correlativity between the total triterpene,oleanolic acid,flavoniods and APX,SOD,CAT,PAL could be significant or extremely significant after high temperature of 45? and low temperature of 4? stress for 4h,and these three secondary product and four stress-tolerant enzymes activities have less correlativity between stressed in these two temperature for short time(1h)and control of non-stressed.