Investigation On PPBI-1 Effecting NO-Induced Cell Death And Flavonoid Production Accumulating In Transgenic Curly Bristlethistle Herb Cells

In higher plants,programmed cell death(PCD,apoptosis)and secondary metabolites synthesis are complex physiological and biochemical function comprising a series of genetically controlled events. It is demonstrated that various stimuli can induce nitric oxide(NO) generation to trigger cell death and enhance secondary metabolites production in plants.Bax inhibitoer-1 is one of new genes which can regulate apoptosis and overexpression of it could not just be able to suppress Bax-mediated programmed cell death but also function as an attenuator of biotic and abiotic types of cell death in model plants. Therefore,we construct transgenic 17-β-Estradiol(Est)-inducible PPBI-1 callus of Curly Bristlethistle Herb via Agrobacterium tumefaciens and study the function of Ppbi-l(a plant Bax inhibitor-1 isolated from Phyllostachys praecox)in reducing NO-mediated cell death and influencing secondary metabolites synthesis to dissect NO signal pathway.The main results are summarized as follows:(1)Agrobacterium tumefaciens-mediated PER8-Ppbi-1 transform callus of Curly Bristlethistle HerbIdentified by Colony PCR and Double enzyme(XhoI and SpeI) digesting,we got the Agrobacterium tumefaciens stain EHA 105 carrying PER8-Ppbi-1.Wide Curly Bristlethistle Herb callus cells was effected by this stain with a certain concentration in 1:250 for 30 minutes in dark, after con-culturing 8 days,killed EHA105 and cultured callus on the selective medium which containing 30mg/L hygromycin.As a result,28 resistant callus cultures were selected.It is determined that Agrobacterium tumefaciens-mediated transformation is an efficient procedure for Curly Bristlethistle Herb(2)Identifying transgenic Ppbi-1 Curly Bristlethistle Herb cellsAfter secondary selections under 80mg/L hygromycin,we got 15 resistant callus cultures,11 of these were positive by PCR analysis which confirmed that the Ppbi-1 gene has been successful integrated into the chromosomal DNA of the transgenic cells.Western blotting analysis indicated that Ppbi-1 gene expression was highly induced by Est.The transgenic Curly Bristlethistle Herb cells overexpressing PPBI-1 protein under a 17-β-Estradiol(Est)-inducible promoter was Successful constructed(3)Establishing suspension culture transgenic Ppbi-1 Curly Bristlet histle Herb cells and Optimizing the culture conditionThe suspension culture of the transgenic cell was initiated from the well-growth transgenic Curly Bristlethistle Herb callus on solid medium, To get the optimum of suspension culture conditions,we studied multiple factors and got the result as:MS medium with hormones(1×10-5M NAA+2×10-6M BA)and 2.5%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 100 rpm,the quantity of inoculation is 3g fresh cell per 50ml medium.Under this conditions,we got dispersible Curly Bristlethistle Herb suspension cell line,then we studied the growth curve of this cell and found the suspension culture cells' cycle is 14days.During culture time,we detected and selected the transgenic Bax cells by PCR.As a result,a stable suspension transgenic cell line of Curly Bristlethistle Herb with high Secondary metabolites content and a high growth speed was Established,which is well prepared for further functional analysis.(4)Overexpression of PPBI-1 reducing NO-mediated cell death and influencing secondary metabolites synthesis in transgenic Curly Bristlethistle Herb cellsNO is a new signal molecule,using sodium nitroprusside(SNP),a donor of NO,pretreated the transgenic suspension cells in the mid-exponential phase of cell growth cycle,we found that 0.5mM SNP can induce suspension cell death,but a reduction in cell death can be observed when is supplemented with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)or Est induced Ppbi-1 overexpression.And the repression of NO-induced cell death with Ppbi-1 overexpression in transgenic lines was apparent even at SNP concentrations up to 1mM.In addition work,we found that most of suspension cells treated with 0.5mM SNP can be stained by Sytox green,some PCD morphologic characteristics such as chromatin condensation and margination can be observed.DNA Ladder was also detected with these cells.As to the control and the cell treated with 0.5mMSNP+20μM Est,the above-mentioned characteristics can't be detected.Furthermore,0.5mMSNP,0.5mMSNP+20μMEst,0.5mM SNP+20μMEst+1mM cPTIO separately treated the end of the mid-exponential phase of cell growth cycle(9th day),12th day harvested suspension transgenic cells,measured the DW and flavonoid concentration,we found that cell treated with 0.5mM SNP+20μM Est+1mM cPTIO had pretty much the same cell growth and flavonoid production as the control;single treated with SNP enhancing flavonoid synthesis,while Est induced Ppbi-1 gene overexpression didn't reduce it. As a result,overexpression of Ppbi-1 gene could reduce NO- mediated PCD,but not secondary metabolites such as flavonoid synthesis which indicated that NO via two different signal pathways induced PCD and enhanced secondary metabolites synthesis in transgenic Curly Bristlethistle Herb cells.