| The bottle-necks of industrialization of using cell culture method for producing secondary metabolite are slow growth and low output of secondary metabolite.The study on mechanism of synthesis pathway of secondary metabolite in medical plant cells is meaningful for solving the problem of low output of secondary metabolite in medical plant cells. Secondary metabolite is the product of medical plant in acclimatization. While medical plants suffer adversity stress,they product secondary metabolite to enhance environment-adapting ability.So it is an important method to improve secondary metabolite content using adversity stress to induce medical plant cells.The double influence of adversity stress in growth and quality of medical plants can not be solved by normal culture and management measures.BI-1 gene is a widely inhibitory factor to apoptosis,it can inhibit programmed cell death(PCD) process mediated by Bax or not.In order to investigate the effect of BI-1 in PCD and secondary metabolite induced by adversity stress,we utilized the transgenic BI-1 Carduus crispus cells,studied the influence of the expression of BI-1 underβ-estradiol-inducible promoter,and discussed the mechanism of synthesis pathway during the above-mentioned process.The main results are summarized as follows:(1) Optimizing the culture condition of suspension culture transgenic BI-1 Carduus crispus cellsThe suspension culture cells from the transgenic BI-1 Carduus crispus callus.To get the optimum of cell growth and flavonoid synthesis, multiple factors were studied,such as different types and concentrations of growth regulators,pH,sucrose concentrations,temperature,and rating speed of the shaker on cells culture.We got the best suspension culture condition:MS medium with hormones(1×10-5 mol·L-1 NAA +2×10-6 mol·L-1 BA) and 3%sucrose,the medium was adjusted to pH 5.8 and then sterilized at 121℃for 20 min before use,the culture temperature is 25℃, shaker's rating speed is 110 rpm,and the suspension culture cells' cycle is 14 days.In order to get the stable transgenic BI-1 Carduus crispus suspension cells line,we detected and selected the transgenic BI-1 cells by PCR.As a result,a stable transgenic BI-1 Carduus crispus suspension cells line with high flavonoid content and a high growth speed was obtained.(2) The PCD and flavonoids synthesize induced by UVAfter treating BI-1 transgenic cells with UV,PCD was observed in induced BI-1 transgenic cells via Sytox green staining,and the apoptosis rate was increasing with time going on.Some morphologic characteristics such as chromatin condensation and Margination appeared about in 36th h,and apoptotic body formation appeared about in 72nd h after UV inducement.DNA Ladder was detected about at the 48th h after inducement.As to the control,the above-mentioned characteristics can't be detected.It indicated that UV can induce PCD in BI-1 transgenic Carduus crispus cells.The cells in different times of the cells culture cycle were treated with UV,and the corresponding flavonoid content was detected at the 14th day,we drew a conclusion,for BI-1 transgenic cells treated by UV at the 11th day,the flavonoid content significantly increased,and the cell fresh weight and total flavonoid yield were,respectively,93.8%and 2.69-fold higher than the cells without treated.It indicated that UV can induce flavonoid synthesis in BI-1 transgenic Carduus crispus cells.(3) NO mediated PCD and flavonoids synthesis under UV stressIn order to study the mechanism of signal transduction of PCD and flavonoid synthesis under UV stress,we detected the NO content.NO burst was observed after treatment with UV,the first increasing was about at the 10th h,and then reached the first phase peak at the 18th h. Consequently,it decreased a little.However,it increased again about at the 32nd h,reached the second phase peak at the 38th h,and higher than the first.NO specific scavenger 2-4-carboxypheny-1-4,4,5,5-tetramethyli-midazoline -1-oxyl-3-oxide(cPTIO) can blocked the NO burst,we found that while NO was blocked,the PCD and flavonoids synthesis induced by UV in BI-1 transgenic cells were suppressed.The result show that NO is the essential factor in PCD and flavonoids synthesis induced by UV in BI-1 transgenic cells.The suppression of NO burst was completed,but flavonoid synthesis was partly,which suggests that NO is not the unique factor in signal transduction of flavonoid synthesis induced by UV in BI-1 transgenic Carduus crispus cells.(4) The infuence of BI-1 in PCD and flavonoid synthesis induced by UV stressWestern blotting analysis indicated that BI-1 gene can be expressed while treated with 17-β-Estradiol(Est,20μmol·L-1).After treated with Est,the BI-1 transgenic cells had no changes in growth and flavonoids content.But while treated the BI-1 transgenic cells with Est before induced by UV stress,the PCD can be suppressed,the flavonoids synthesis and NO content can not be.The results suggest that there are two different signal transductions for PCD and flavonoid synthesis in BI-1 transgenic Carduus crispus cells induced by UV stress. All the results suggest that:(1) The PCD and flavonoids synthesize induced by UV;(2) NO involved in the processes of signal transductions of PCD and flavonoid synthesize in BI-1 transgenic Carduus crispus cells induced by UV stress;(3) NO is the sharing signal in PCD and flavonoid synthesize in BI-1 transgenic Carduus crispus cells induced by UV stress, but there are two different pathway in NO downstream through that UV can induce PCD and flavonoids synthesize respectively.And the specific BI-1 locus is in the signal transgenic pathway of PCD.All the results provide the theoretical basis for the molecular regulation of BI-1 gene in growth and quality of medical plants,and now the correlation research is still going on. |
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