【作者】 王世华；

【导师】 沈振国；

【作者基本信息】 南京农业大学 ， 植物学， 2007， 博士

【摘要】 重金属在土壤中的累积、迁移和形态转化,导致土壤环境质量恶化,严重影响土壤圈的物质循环,并通过食物链危害人类的生命和健康。硅是自然界中非常丰富的元素,对处于逆境胁迫的植物具有特殊的营养生理功能。本研究拟采用纳米硅制剂作为硅源,尝试寻找一种更方便、可行、有效的利用硅的方法,并研究纳米硅制剂增强水稻抗重金属毒害的机理。采用盆栽实验比较了6个品种水稻在重金属复合污染条件下的生长情况和重金属积累差异以及叶面喷施纳米硅制剂的影响。在重金属Cd（5 mg/kg）、Pb（200 mg/kg）、Cu（250 mg/kg）和Zn（300 mg/kg）复合污染下,在水稻的不同生育期內（苗期、分蘖期、抽穗期）叶面喷施硅酸钠和正硅酸乙酯配制的纳米硅制剂（2.5 mM）,促进了6个品种水稻的生长发育,增加了水稻地上部生物量,对常规稻的百粒重、单株有效穗数影响显著（P＜0.05）,叶面喷施纳米有机硅也显著增加杂交稻地上部生物量。无论是否叶面喷施纳米硅,四种金属元素的籽粒吸收系数顺序基本上表现为Cd最高,Pb最低,Cu、Zn居中,显示Cd具有较强的向籽粒迁移的能力。对重金属在水稻籽粒的吸收能力,不同品种间存在显著差异（P＜0.05）.叶面喷施硅制剂后,所有品种水稻重金属的吸收系数均趋向下降,在杂交稻中的下降幅度大于常规稻,叶面喷施有机硅的效果好于无机硅。在较高污染水平下,叶面喷施两种2.5 mM纳米硅制剂,显著提高“优优128”的水稻百粒重及单株穗重（P＜0.05）,并且显著降低籽粒中Cd、Pb、Cu、Zn的吸收量。随着Cd处理浓度的增高,叶面喷施硅对重金属在籽粒中积累的抑制效应越显著（P＜0.05）。喷施较高浓度（5和10 mM）纳米硅有利于水稻的生长,对粒重和生物量的促进效应达显著水平（P＜0.05）。10 mM以下的无机硅和有机硅配制的纳米硅制剂抑制水稻对重金属的吸收表现出浓度效应。同一浓度下,喷施有机硅源的纳米硅比无机硅源纳米硅的促进作用更大。通过水培实验,研究了叶面喷施纳米无机硅制剂（2.5 mM）对20μM镉胁迫处理14d的水稻幼苗营养生长、光合特性、质外体运输以及处理3d和14d后抗氧化系统的影响,并利用RT-PCR技术探讨了纳米硅制剂对镉处理3d水稻幼苗γ-谷氨酰半胱氨酸合成酶（γ-ECS）基因表达的影响。结果显示,叶面施硅可以改善镉胁迫所导致的水稻养分不平衡,改善水稻的生长。使水稻幼苗的生物量增加22%,株高增长8%,根系长度增长14%。叶面喷施纳米硅降低了Ca在水稻地上部的分布,使Cd胁迫植株的Ca在地上部的浓度恢复到对照水平。叶面施硅提高了Mg在水稻幼苗体内的浓度。在微量元素中,叶面施硅显著提高Cd胁迫的幼苗地上部zn、Fe和Cu浓度（P＜0.05）。叶面喷施纳米硅降低了水稻幼苗对镉的吸收,并降低了其在地上部分布的比例;施硅处理的水稻幼苗木质部汁液中镉的浓度降低了32.7%。通过膜不浸透的荧光染料PTS（tri-sodiunl-8-hydfoxy-1,3,6-pyrenesulpbonate）的吸收实验证实,施硅可以减少水稻幼苗对PTS的吸收量,说明质外体运输路径通透性变小是施硅使地上部镉积累减少的原因之一。叶面喷施纳米硅有提高水稻幼苗净光合速率（Pn）、降低水稻幼苗蒸腾速率（Tr）的作用,但与对照相比差异不显著。在2QμM Cd存在下,施硅可显著缓解镉胁迫引起的过度蒸腾,提高了水稻幼苗对水分的利用率（WUE）。镉胁迫提高了水稻叶片的气孔导度,但叶肉细胞的光合活性降低,胞间CO₂浓度（Ci）升高;叶面喷施纳米硅增加了水稻幼苗叶肉细胞的光合活性,提高了水稻幼苗对CO₂利用率,使净光合速率Pn升高。叶面喷施纳米硅显著降低了镉胁迫引起的膜质过氧化程度（P＜0.05）,随处理时间延长,丙二醛（MDA）含量显著下降。叶面喷施纳米硅使水稻幼苗中还原型AsA含量比对照显著提高（P＜0.05）;总的AsA含量也表现为上升趋势;地上部AsA含量明显高于根系中含量。在镉处理后,水稻幼苗中总谷胱甘肽（GSH+GSSG）含量较对照显著下降（P＜0.05）。而氧化型谷胱甘肽（GSSG）随处理时间延长显著增加（P＜0.05）,施硅后,总谷胱甘肽在水稻幼苗的地上部和根系含量均显著增加,氧化型谷胱甘肽随处理时间延长显著下降（P＜0.05）,而且GSH/GSSG在施用纳米硅后比例上升。叶面喷施纳米硅提高GR活性,且随时间延长,可以对镉胁迫引起的GR活性下降有显著缓解作用。叶面施硅初期抑制APX活性,随处理时间延长,APX活性比对照略有上升,但对镉胁迫下幼苗影响不显著。叶面喷施纳米硅后,SOD活性表现表现为先下降后上升,根系中的上升幅度要大于地上部。叶面施硅也提高了水稻POD活性,根系中的POD活性高于地上部。无论是否喷施纳米硅,CAT活性先降后升,但后期活性降低,地上部高于根系,说明叶面喷施纳米硅提高了水稻幼苗的抗氧化能力。镉胁迫使水稻幼苗的NPT含量上升（P＜0.05）,但增加幅度小于施硅对幼苗体内NPT含量的影响。利用半定量RT-PCR技术以及特异引物,扩增了水稻品种“优优128”根和叶片中γ-谷氨酰半胱氨酸合成酶（γ-ECS）基因转录产物。扩增片段经序列测定以及通过基因Bank Blast相似性检测分析,证实上述基因片段序列与水稻的gamma-glutamylCysteine synthctase基因序列具有很高的同源性,为99%。结果表明:Cd胁迫可以诱导水稻幼苗根部和叶片中γ-ECs基因的过量表达,而且这种诱导在根部更为显著,而叶面喷施纳米硅对γ-ECS基因表达的诱导作用说明叶面喷施纳米硅有利于水稻幼苗合成GSH提高对镉毒害的抗性。更多还原

【Abstract】 The contamination of soils with metals is a major environmental problem throughout the world.Soils polluted with metals may threaten ecosystems and human health.Silicon is the second most abundant element on the surface of the earth,and there is increasing evidence that Si has a number of beneficial effects on plant growth under biotic and abiotic stresses.The crossing of inorganic chemistry,environmental chemistry,material chemistry and life science has formed a new study field to utilize environment matter and bio-nanometer matter in agriculture.In the present study,we try to use different nanometer silicon preparation sprayed onto the leaves of rice(Oryza sativa L.) under heavy metal stress,to find a efficient method in the utilize of silicon to reduce the uptake of heavy metal, and discover the mechanism.In pot experiment 1,six rice cultivars were planted in the soil artificially amended with multi-metals(Cd,5 mg/kg;Pb,200 mg/kg;Cu,250 mg/kg,Zn,300 mg/kg),and two kinds of nanometer Si preparation(from TEOS and sodium silicate were foliarly applied to investigate their effects on the growth of rice and heavy metals uptake in rice grains.Foliar application of nanometer silicon preparation can improve the growth of rice,significantly increase the biomass,weight of per 100 grains and spike number of per plant.The distribution of heavy metal in rice was root＞shoot＞grain.The uptake coefficient of heavy metal is Cd＞Zn＞Cu＞Pb whether with or without silicon.Hybridize cultivars accumulated more Cd in grains than that of general cultivars but there is little difference between hybridize cultivars and general cultivars on the accumulation of Zn and Cu.Foliar application of nanometer silicon reduced the accumulation of all heavy metals and effect was more pronounced on hybride than on general cultivars.Pot experiment 2 was conducted to investigate the effects of foliar application of Si on heavy metals uptake in rice(cv.youyou 128) grain under Cd,Pb,Cu and Zn stress.Foliar. application of Si alleviated the toxicity of heavy metals on rice.Compared with application of inorganic Si,the application of organic Si was more positive in alleviating heavy metal toxicity.With the application of Si,the weight of per 100 grains,and weight of per spike were increased significantly(P＜0.05),the accumulation of Cd,Pb,Cu,Zn in the grain were decreased significantly(P＜0.05).The uptake coefficient and the accumulation of heavy metals in grains were also decreased.With the increasing of Cd concentration in soil,the alleviating of heavy metal toxicity by Si application increased.These results indicated that foliar application of Si was a propriety method of alleviating heavy metal toxicity and decreasing metals accumulation in rice.Foliar application of nanometer silicon at 5 mM and 10 mM significantly increased the weight of per 100 grains and biomass were significantly increased.The uptake of heavy metal decreased with increasing application rate of nanometer silicon preparation.The content of Zn and Pb was lower than the threshold concentrations for safely edible food. The application of Si reduced Cu concentration,which was lower than the safely edible threshold.But the Cd concentrion was slightly higher than the safely edible threshold.At the same application rate,organic nanometer silicon was more effective than inorganic nanometer silicon for improving rice growth and decreasing heavy metal accumulation.In the present study,solution culture experiments were also conducted to investigate the influence of 2.5 mM nanometer silicon preparation on growth of rice seedlings under 20μM Cd stress.The results showed that.Si application significantly alleviated the toxicity of Cd in rice.Si reduced the distribution of Ca in the shoots,increased the Mg,Zn,Fe and Cu concentration(P＜0.05),increased chlorophyll content,promoted the growth of rice and increased the biomass of the plants.Application of Si increased Pn and WUE,but decreased Tr,Gs and Ci in leaves of rice seedlings under Cd stress.Si decreased Cd content in the shoots and roots of rice.The concentration of Cd in xylem and the translocation of Cd from roots to shoots were decreased by Si application. The results of applastic fluorescence tracer PTS(trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid) showed the significant reduction of apoplastic transport in nanometer silicon preparation applied plants,and the reduction of fluorescence was more than that of Tr reduced by the application of nanometer silicon,this suggested that Si restrained the apoplastic transport of Cd and alleviated the toxicity of Cd.Si decreased the content of MDA content and affected the activity of activities of SOD, POD,CAT,APX and GR.Activity of SOD in shoots was increased under short-time Cd stress,but decreased later.Activity of SOD in roots was decreased under short-time Cd stress,but increased later.The application of silicon decreased the activity of SOD.Cd stress and silicon increased activity of POD.The activity of POD in roots was higher than that in shoots.Activity of CAT in shoots was increased under short-time Cd stress,but decreased later.Application of silicon increased the activity of CAT in both of shoots and roots.Activity of GR was decreased by Cd,but was increased by silicon.Activity of APX was little affected by Cd or silicon.Total ascorbate,total GSH,the ration of GSH/GSSG was increased by the application of silicon.NPT content was increased in the nanometer silicon preparation applied rice seedlings. So transcription amount for the key enzyme regulating GSH production(γ-ECS) was detected and the result showed that transcription amount forγ-ECS was increased both under Cd stress and silicon application.GSH played a role in Cd tolerance and silicon increased GSH production.更多还原