【作者】 潘建伟；

【导师】 朱睦元；

【作者基本信息】 浙江大学 ， 遗传学， 2002， 博士

【摘要】 铝毒是酸性土壤影响作物生长的主要原因，已成为植物胁迫生理学与遗传学的重要研究内容。然而，目前对植物铝毒机制仍不清楚。大麦是铝毒极为敏感的作物，本实验以大麦为材料，开展了铝毒诱导的根尖细胞超微弱发光、姐妹染色单体交换(SCE)、细胞程序性死亡、耐铝基因工程及边缘细胞在铝毒中的功能研究。试图为植物铝毒及耐铝机制和遗传改良研究探索新途径。 利用超微弱发光鉴定植物根尖铝毒及耐铝性，目前国内外仍未见正式报道。本实验表明，利用根相对伸长率可将30个大麦品种划分为耐铝、中等耐铝和敏感三种类型；而利用根尖相对发光率可将其分成高耐铝、耐铝、中性、敏感和高敏感五种类型。根相对伸长率与根尖相对发光率之间具有正相关性(r=0.63295)。并且根尖相对发光率比根相对伸长率更为灵敏。建立的大麦根尖超微弱发光技术用于鉴定大麦耐铝性是完全可行的。 首次研究了铝毒诱导植物SCE的效应。观察到用20μmol／L Al诱导敏感大麦cv．2000-2的SCE率比耐铝大麦cv．Humai16高，铝毒诱导的SCE率与大麦耐铝性呈负相。经不同铝浓度处理，这两个品种的SCE率具有相类似的变化曲线，但两者的临界浓度(诱导SCE率最高的浓度)显著不同(分别为10和320μmol／L)。这些结果表明，大麦根尖SCE对铝毒反应极为敏感，可作为植物铝毒研究的敏感指标。用于鉴定大麦耐铝性。进一步的实验表明，Vc能明显抑制铝毒诱导的SCE。推测铝毒很可能通过诱导ROS间接诱导SCE的发生。 铝在根尖细胞壁上的积累是铝对植物根尖产生毒害的先决条件。通过对耐铝性差异极其显著(P＜0.01)的大麦cv．2000-2与cv．Humai16铝处理后的Morin荧光检测，观察到cv．2000-2根尖荧光明显比cv．Humai16强，表明敏感品种根尖细胞壁上铝积累比耐铝品种多。同时，铝处理4h的cv．2000-2根尖果胶甲基酯酶PME活性显著上升，但处理24h后PME活性又显著下降；而cv．Humai 16的PME活性无显著性差异。表明细胞壁上铝积累与PME活性变化有密切相关性，PME活性在铝毒敏感性中可能起着重要作用。 本研究首次报道了铝毒诱导大麦根尖细胞程序性死亡。在铝胁迫或无铝恢复培养过程中产生的细胞伸长或生长的不可逆抑制起源于细胞死亡。0.1-5.0mmol/L铝处理大麦根尖8h或悬浮细胞24h均产生了梯状DNA，但未观察到凋亡小体的形成。故将这种细胞死亡形式称为铝诱导PCD。超微弱发光的结果表明，铝诱导PCD很可能是通过ROS信号传导途径来实现的。铝毒诱导的不可逆抑制表明，一旦细胞死亡程序被激活后，无论铝存在与否，细胞死亡将成为一个不可逆的过程。 通过基因工程方法导入凋亡抑制基因是作物抗逆性遗传改良的一条新策略，也是细胞凋亡研究成果的具体应用，目前国内仍未见正式报道。本研究试图通过农杆菌介导法将凋亡抑制基因cedg导入烟草，获得高耐铝性的转基因植株。结果表明，利用pGEMJ松八载体上的Sacl和Xbal位点，将pBS载体上的cedg基因成功地克隆到表达载体pBllZI（含 CaMV 355）上，定名为 pBI－ced习。通过农杆菌感染烟草叶盘，经筛选、诱导和分化后，共获得96株再生植株厂。卜经PCR检测和h 杂交表明，目的基因cedg已整合到烟草基因组中。有关转基因植株的CCdg表达活性及其对生长发育的影响、耐铝性鉴定将在T；、TZ、T。代进行。 根边缘细胞（BC）研究是最近几年植物学和遗传学的新领域之一。本实验表明，大麦 cv．Hang 98 BC发育启动与根发育几乎同步，这与豆科植物 BC发育不同。在悬空气培中，大麦BC发育属于非组成型表达的过程。在水培中，每个根每天仍释放300～350个BC，属于组成型表达过程。根冠PME活性检测表明，大麦BC发育与PME活性具有密切的相关性，PME活性在BC游离过程中起着重要作用。温度对大麦根伸长有明显的抑制作用，但对BC发育影响不大。 铝毒对离体收集的 BC活性影响非常明显。大麦耐铝品种 cv．Humall6的 BC经 20和 50 v moffe AI处理后，其活性无显著性差异，而敏感品种 cv．2000．2的BC活性却受到显著抑制，说明BC的耐铝性与其个体植株水平的耐铝性相一致。到目前为止，国内外有关铝毒对植物BC发育的影响仍未见正式报道。本实验表明，铝毒对大麦 BC发育有明显的抑制作用。20 u mow AI对。2000上的 BC发育有显著性抑制o＜0刀5卜但对cv．Humal 16的BC发育的影响不大j0 u mowAI对这两个品种的BC发育均有显著性抑制0m．of卜PME活性检测表明，铝毒很可能通过抑制根冠细胞壁PME活性从而抑制BC发育。另外，根据我们的实验，BC及其粘液对铝毒诱导的大麦根伸长抑制的保护功能很小。但在低浓度铝或短时间胁迫下，BC及其粘液对其根尖分生组织和根冠分生组织具有一定的保护作用。这种保护作用可能与粘液中含有糖醛酸有关。更多还原

【Abstract】 Al toxicity is a major limited factor for crop production and quality in acid soil, and it has become the focus of ongoing research in the area of plant stress physiology and genetics. At present, however, Al toxicity mechanisms in plant are still not clearly understood. Barley, which was used as experimental material in this study, is one of the most Al-sensitive crops. Al toxicity-induced ultraweak luminescence (UL), sister-chromatid exchanges (SCEs), programmed cell death (PCD) in root tips of barley, Al-resistant genetic engineering and the role of root border cells in Al toxicity were studied respectively, in order to explore some new pathways for Al toxicity, Al-resistant mechanism and genetic improvement.The determination of Al toxicity and Al-resistant ability in plant root-tip using UL is still not reported. Our experiments show that, according to root relative elongation rates (RERs), 30 barley cultivars can be divided into three types such as Al-resistant cultivars, intermediate cultivars, and Al-sensitive cultivars. However, according to root tip relative UL rates (RULRs), these cultivars can be divided into five types such as highly Al-resistant cultivars, Al-resistant cultivars, intermediate cultivars, Al-sensitive cultivars, and highly Al-sensitive cultivars. These results suggest that RULRs as an Al toxicity indicator is more sensitive than RERs. There was a statistically significant and weak positive correlation between RERs and RULRs (r=0.63295), indicating that it is possible to estimate barley Al-resistant ability using UL.Al-induced sister-chromatid exchanges (SCEs) were first time determined. Our experiments show that Al-induced SCE rates are higher in cv. 2000-2 than in cv. Humai 16 at 20 umol/L Al level, indicating that there is a negative correlation between Al-induced SCEs and Al-resistant ability in barley. Under different Al concentrations, there are same change curves of SCE rates hi cv. 2000-2 and cv. Humai 16, but their critical concentrations, which can induce the highest SCE rates, were significantly different from each other. Critical concentrations of cv. 2000-2 and cv. Humai 16 are 10 and 320 umol/L Al respectively. These results suggest that SCEs are very sensitive to Al toxicity hi the root tips of barley and can be also served as a sensitive indicator in Al phytotoxicity. The Al-resistant ability of barley may be assessed by their critical concentrations. Further studies show Al toxicity indirectly induces SCEs through inducing ROS.Al accumulation on cell wall of root tips is the primary prerequisite of toxic effect of Al on root tips in plants. According their RERs, there is a statistically significant difference (PO.01) between cv. 2000-2 and cv. Humai 16. Morin fluorescent staining shows that fluorescence in the root tips of cv. 2000-2 is more intense than that in the root tips of cv. Humai 16, indicating that more Al is accumulated on the root tips of cv. 2000-2 than cv. Humai 16. 4-h Al treatment significantly enhanced PME activity hi root tips of cv. 2000-2 and after 24 h Al treatment, PME activity significantly decreased. However, PME activity did not show significant changes in cv. Humai 16. These results suggest that Al accumulation on cell wall is very closely related to PME activity on the root tips, and PME activity has an important role in Al toxic sensitivity.We first reported Al-induced programmed cell death in the root-tip cells of barley. During Al treatment or recovery culture without Al, the irreversible inhibition of cell elongation or growth was resulted from cell death. 0.1-5.0 mmol/L Al can induce DNA ladders in the root-tips (treated for 8 h) and suspension cells (treated for 24 h) of barley, but no apoptotic bodies were observed. Therefore, cell death caused by Al is called as Al-induced PCD. The results of UL show Al induced PCD possibly via a ROS-activated signal transduction pathway. Irreversible inhibition induced by Al show that cells were doomed to death whether Al stress or not, once the cell death program was activated in Al-t更多还原