【作者】 Alieu Mohamed Bah（艾留）；

【导师】 邬飞波；

【作者基本信息】 浙江大学 ， 作物栽培与耕作学， 2011， 博士

【摘要】 土壤重金属污染严重影响农产品品质和产量,甚至沿食物链富积威胁到人畜健康。为寻求更有效、可行的治理方法,近年来,一种针对重金属污染的植物修复技术(phytoremediation)引起了公众及科学界的广泛兴趣,即通过金属积累(metal-accumulating)植物吸收、转运并积累从而去除土壤中有害金属(包括放射性物质)。经野外调查,我们在浙江东南部地区受电镀废水、废弃物严重污染的土壤上发现了一种无重金属中毒症状、生长旺盛、生物量大的多年生香蒲科(Typhaceae)香蒲属植物一狭叶香蒲(Typha angustifolia L.)。本研究采用盆栽试验,旨在探讨该狭叶香蒲对对铬、镉、铅胁迫的反应及耐重金属毒害的生理机理与耐性相关特异蛋白。主要研究结果如下：盆栽试验研究了香蒲对铬、镉、铅毒害的耐性,并分析测定了活性氧代谢等相应的生理反应指标。狭叶香蒲播种后90 d和130 d(记作D90,D130)分别进行1mM Cr(Ⅵ)、1mM Cd、1mM Pb胁迫处理(denoted as Cr-D90、Cd-D90、Pb-D90和Cr-D130、Cd-D130、Pb-D130),以不施重金属为对照(Control-D90、Control-D130)。结果表明,污染环境生态型狭叶香蒲对重金属毒害具有很强的抗性,各种重金属胁迫下香蒲植株地上部并未表现出失绿或坏死等中毒症状；铬、镉、铅处理分别显著增加狭叶香蒲根和地上部茎叶中铬、镉、铅含量,此外,铬处理(Cr-D90和Cr-D130)茎叶镉、锰含量比对照显著增加26%、113%和38%、782%,明显降低铅浓度,但对铜、锌、钙浓度无显著影响。镉处理(Cd-D90和Cd-D130)显着增加茎叶中钙、锰含量,明显降低铬、锌浓度,对铜、铁浓度无显著影响。铅处理(特别是Pb-D130处理)显著降低锰和镉含量。铬、铅、镉胁迫诱导植株体内超氧物歧化酶SOD (superoxide dismutase)、过氧化物酶POD (peroxidase)等抗氧化酶活性的提高；铅胁迫还显著促进过氧化氢酶CAT(Catalase)、谷胱甘肽过氧化物酶GPX(Glutathione peroxidase)、双脱氢抗坏血酸还原酶DHAR(Dehydroascorbate reductase)、抗坏血酸过氧化物酶APX(Ascobate peroxidase)等抗氧化酶活性的提高。研究了香蒲对铬、镉、铅毒害耐性相关的特异蛋白。播种后90d和130d香蒲苗分别进行1mM Cr(Ⅵ)、1mMCd、1mM Pb胁迫处理30d,取植株茎叶,提取蛋白样品,应用蛋白双向电泳与质谱分析,鉴定铬、镉、铅胁迫特异表达蛋白。双向电泳每凝胶图谱检测到约1600多个蛋白点。铬、镉、铅处理下,与对照相比,Cr-D90、Cd-D90、Pb-D90(?)(?)Cr-D130、Cd-D130、Pb-D130分别检测到44、46、66个和33、26和62个差异表达蛋白点;其中3、1和8个差异表达蛋白点在二种幼苗(D90、D130)中同时表现胁迫处理条件下上调表达：5、8、5个差异表达蛋白点在D90、D130其中之一幼苗表现胁迫处理上调表达[Fold increase (treated/control)≥3]。将差异表达的目标蛋白质点切下,经脱水、还原、烷基化、酶解和脱盐等处理,MALDI-TOF-MS质谱分析,进一步应用生物信息学技术,分别鉴定到22个和4个重金属胁迫上调和下调表达蛋白点。铬胁迫诱导ATP合成酶、核酮糖二磷酸羧化酶-加氧酶小亚基和类卟啉原-Ⅲ氧化酶的表达：镉胁迫诱导核酮糖二磷酸羧化酶-加氧酶大亚基表达：铅胁迫促进碳水化合物代谢途径中果糖激酶活性,提高了核酮糖二磷酸羧化酶-加氧酶大亚基以及镁原卟啉Ⅸ螯合酶活性。铬、铅处理抑制e1F4F类蛋白表达,镉和铅阻碍了叶绿体FtsZ类蛋白和GF14omega表达。另外,我们观察了铬、镉、铅胁迫对香蒲超微结构的影响。铬、镉、铅胁迫损害了香蒲超微结构,铬、镉、铅处理的幼苗显示了扩大的液泡和粗糙的内质网膜,叶绿体减少。更多还原

【Abstract】 Heavy metal pollution in soils poses a major environmental hazard to terrestrial plants and ultimately metal toxicity to animals and humans via food chain. It is crucially important to develop cost-effective remediation strategies for these metals. One of such emerging technology is phytoremediation through the use of green plants to extract, sequester and detoxify the pollutants. We found a kind of gramineous plant species, Typha angustifolia, with high resistance to heavy metal stress. The present research was conducted to evaluate its ability to tolerant Cr, Cd and Pb stress and the relevant physiological responses, and to identify stress-specific protein for Cr, Cd and Pb toxicity tolerance in T. angustifolia. The results were as follows:Pot experiments were conducted to study the potential of T. angustifolia plants for Cr, Cd and Pb toxicity tolerance and subsequent response in antioxidant defence system after 30 d exposure of 1 mM Cr, Cd or Pb. Typha angustifolia showed high tolerance to heavy metal toxicity with no visual toxic symptom when exposed to metal stress, and Cd/Pb addition also increased plant height and biomass especially in Pb treatment. Along with increased Cr, Cd and Pb uptake in metal treatments, there was enhanced uptake of plant nutrients including Ca and Fe; and Zn in Pb-treatment. A significant increase in MDA content, SOD and POD activities were recorded in plants subjected to Cr, Cd or Pb stress. Furthermore, Pb-stress also improved CAT, APX and GPX activities; whereas Cr-stress depressed APX and GPX. The results indicate that enzymatic antioxidants and Ca/Fe uptake were important for heavy metal detoxification in T. angustifolia, stimulated antioxidative enzymes and Ca, Fe and Zn uptake could partially explain its hyper-Pb-tolerance.Typha angustifolia of 90 (D90) and 130 d (D130) old plants were subjected to 1 mM Cr, Cd and Pb and samples were collected 30 d after treatment.2-DE coupled with MS (mass spectrometry) was used to analyze and identify Cr, Cd and Pb-responsive proteins. More than 1600 protein spots were reproducibly detected on each gel. wherein 44,46,66 and 33,26,62 spots in D90 and D130 samples were differentially expressed by Cr, Cd, Pb over the control, respectively. Of these differentially expressed proteins,3,1,8 overlapped in D90 and D130; while 5,8,5 with regulation factors above 3 in one of D90 or D130 samples. Total of 22 and 4 up-and down-regulated proteins were identified using MS and data bank analysis. Cr induced expression of ATP synthase, RuBisCO small subunit and coproporphyrinogenⅢoxidase; Cd induced RuBisCO large subunit; Pb up-regulated carbohydrate metabolic pathway enzymes of fructokinase, and improved RuBisCO activase and large subunit, Mg-protoporphyrinⅨchelatase. Contrarily, elF4F was inhibited by Cr/Pb, chloroplast FtsZ-like protein and GF14omega impeded by Cd and Pb respectively.Furthermore, we investigated ultrastructure of T. angustifolia in response to Cr, Cd and Pb stress. Cr, Cd and Pb toxicity stress distort the ultrastructure and the treated seedlings showed that the vacuoles were expanded and endoplasmic membrane appears to be rough. The levels of chloroplast in the plant cell were visible and prominent, yet the heavy metal stress caused an imbalance.更多还原