【作者】 王秀莲；

【导师】 徐茂军；

【作者基本信息】 浙江工商大学 ， 生物化工， 2008， 硕士

【摘要】 高等植物细胞程序性死亡（细胞凋亡）和次生代谢产物合成积累是受细胞内部相关基因调控的复杂生理生化活动,研究表明外界刺激因子可以通过产生NO等信号分子介导细胞凋亡和促进次生代谢产物合成。Bax inhibitor-1（bi-1）是一个新型的凋亡调控基因,它可以抑制由Bax以及生物、非生物因素引起的模式植物细胞死亡。因此,为探讨NO是否依赖相同的信号途径诱发细胞死亡和次生代谢物质合成,本文通过农杆菌介导法构建雌二醇诱导型Ppbi-1（紫竹中克隆鉴定而得）基因工程飞廉细胞株,获得稳定表达的转基因细胞后,研究了Pp-bi-1基因表达对NO诱发的飞廉细胞死亡和次生代谢产物合成的影响,初步探讨了NO介导的信号转导机制。主要实验结果如下:（1）农杆菌介导的PER8-Ppbi-1质粒转化飞廉愈伤组织细胞通过菌落PCR、回转酶切筛选出阳性的PER8-Pp-bi-1/EHA105农杆菌菌株,以1:250的菌液浓度,暗处感染飞廉愈伤组织细胞30min,共培养后以30mg/L的Hyg作为选择浓度筛选转化细胞,在12皿细胞中筛选获得28个抗性细胞系。这说明,农杆菌介导的共培养转化法适用于飞廉愈伤细胞。（2）转诱导型Ppbi-1基因飞廉细胞鉴定对获得的28个抗性细胞系,通过80mg/L的Hyg再次筛选,得到15个抗性细胞系,进一步的PCR分子鉴定,得到11个转基因细胞系,PCR检测和Western blotting检测的结果表明,外源Ppbi-1基因已成功转入飞廉的愈伤组织细胞基因组中,并在20μmol·L^-1雌二醇（Est）诱导下能够稳定表达。（3）飞廉转基因细胞悬浮系的建立及培养条件优化在固体培养的转Ppbi-1基因飞廉细胞中,挑选健康的愈伤细胞接种到MS液体培养基,为使转基因细胞能够更好生长,对液体培养基及培养条件进行了优化,得到最适的培养条件为:以pH 5.8的MS+1×10-5MNAA+2×10-6MBA+25g/L蔗糖为培养基,培养温度25℃,摇床转速100rpm,接种量为3g/50ml培养基;以此培养条件培养细胞,能得到分散、均一且生长迅速的悬浮细胞系;测定细胞生长曲线,发现该悬浮培养细胞的生长周期为14天;细胞悬浮培养期间,定期的PCR检测,保证了转基因细胞的稳定性,避免了培养中出现的一些假阳性细胞,为进一步实验提供了可靠的实验材料。（4）Ppbi-1基因表达对NO诱发的飞廉细胞死亡和次生代谢产物合成的影响NO是一个新型信号分子,可以诱导植物细胞死亡和促进次生代谢产物合成。本实验中,利用NO供体SNP处理处于对数生长期转基因飞廉悬浮细胞,发现在0.5mM浓度处理下细胞出现褐化死亡,NO专一性淬灭剂cPTIO和20μmol·L^-1Est诱导外源基因bi-1表达均可明显抑制由0.5mM SNP引起的细胞死亡,进一步研究发现,Ppbi-1基因能抑制1mMSNP引起的细胞死亡。对0.5mM SNP和0.5mM SNP+20μM Est处理的飞廉转基因细胞培养三天后用特异性核染料sytox染色,0.5mMSNP单独处理的细胞大部分核被染色,而且细胞核出现了核凝聚、以及染色质边集等凋亡细胞的特征;提取细胞DNA电泳分析,观察到了DNA LADDER,这说明,该细胞可能已经发生凋亡,而Est诱导Ppbi-1基因表达的细胞均未出现这些凋亡特征。以0.5mMSNP、0.5mMSNP+20μMEst、0.5mM SNP+20μMEst+1mM cPTIO处理处于对数生长末期（第9天）的细胞,第12天收获细胞,测三组细胞的生物量和总黄酮含量,SNP单独处理,飞廉黄酮类次生代谢产物合成加强,Est诱导Ppbi-1基因表达,不会抑制NO对黄酮类次生产物的合成加强作用,而添加cPTIO后明显抑制该产物的合成。实验结果表明,Ppbi-1基因可以特异性的抑制由NO介导飞廉细胞凋亡,但对NO诱发飞廉细胞次生产物的合成积累无抑制作用,由此推测,NO可能通过两种不同机制诱导飞廉细胞凋亡和促进次生代谢产物合成。更多还原

【Abstract】 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.更多还原