【作者】 汪芳；

【导师】 邬飞波；

【作者基本信息】 浙江大学 ， 作物学， 2008， 硕士

【摘要】 镉（Cadmium,Cd）是毒性最强和农田受污染最普遍的重金属之一,土壤镉污染不仅严重影响作物的产量和品质,并可通过食物链富集危害人体健康。对于已污染土壤,虽然曾提出过包括利用超积累植物进行生物修复降低土壤重金属含量等多种方法,但迄今均未能有效地应用于大面积土壤的治理;对于中、轻度大面积污染的农田,筛选耐镉且食用器官低积累作物品种和改进农艺与化学调控技术缓解镉毒、减少作物镉吸收与积累,是有效利用自然资源和保证农产品安全生产的重要途径。植物螯合肽PCs是植物细胞内螯合镉离子,缓解镉毒性的重要物质,而谷胱甘肽（GSH）是PCs合成前体,因此,探讨外源GSH对植物镉毒害的影响具有重要的理论与实践指导意义。本研究在筛选耐镉性不同大麦基因型的基础上,探讨了不同耐镉性基因型在镉胁迫下添加外源GSH对镉胁迫大麦生长、养分吸收、光合特性、细胞超微结构和活性氧代谢等的影响及基因型差异,旨在探讨外源GSH缓解大麦镉毒害及基因型差异的细胞生理与生化机制。主要研究结果如下:1.选取不同类型与生长习性大麦基因型105份,两次连续的水培镉胁迫试验,分析测定SPAD值、株高、生物学产量等,筛选大麦耐镉基因型。结果表明,20μM Cd处理,显著降低大麦叶片SPAD值、株高、根长与体积、地上部和根系干重,显著抑制Zn、Mn和Cu的吸收和转移。基因型间差异显著,其中萎缩不知和吉啤1号的上述生长与生理指标在两次试验中下降幅度最小;而东17和苏引麦2号下降幅度最大,且植株镉毒害症状最严重。通过试验分别筛选获得耐镉和镉敏感基因型萎缩不知和东17。此外,植株体内镉含量也存在显著的基因型差异,参试品种中以萎缩不知含量最高,吉啤1号最低。2.以筛选所得镉耐性基因型萎缩不知和敏感基因型东17为材料,水培试验,设对照（基本培养液）、GSH(20 mg L^-1GSH)、Cd（5μM Cd）和Cd+GSH(5μM Cd+20 mg L^-1GSH)4个处理,研究外源GSH对镉胁迫下大麦生长和营养元素吸收和分配的影响及基因型差异。结果表明,5μM Cd处理下,地上部镉含量,萎缩不知显著高于东17,但东17的镉毒害症状却更为严重;两个基因型Zn、Mn和Cu的吸收均受到显著抑制。镉胁迫条件下添加20 mg L^-1GSH（Cd+GSH）显著减少大麦幼苗对镉的吸收和积累;但显著提高植株对Zn、Mn和Cu的吸收,其含量甚至超过对照水平;显著缓解大麦幼苗镉毒害症状,其中尤以敏感基因型更明显,其株高、根长和干物质积累较5μM Cd处理增加幅度均高于耐性基因型。3.研究了外源GSH对镉胁迫下大麦细胞结构和光合特性的影响及基因型差异。结果表明,镉胁迫显著降低两个基因型的叶绿素含量及光合作用能力;叶绿体基质和基粒片层出现肿胀,基粒垛叠松散,甚至有些片层发生降解,嗜锇颗粒增加,淀粉粒数量减少,在镉敏感基因型东17中表现更为严重;根细胞核膜不规则凹陷,液泡数量显著下降;敏感基因型中质体和线粒体的脊数量显著减少,而耐性基因型萎缩不知根细胞在镉胁迫下淀粉粒在质体中累积。这种细胞结构变化的基因型差异可以部分说明它们的耐镉性差异。Cd+GSH处理,两个基因型叶绿体中淀粉粒数量增加,嗜锇颗粒显著减少;叶绿素含量、光合速率、PSⅡ光化学效率、气孔导度以及蒸腾速率都较Cd处理显著上升。东17根细胞中由镉胁迫引起的线粒体和质体的损伤得到改善,细胞核基本恢复正常。4.研究了外源GSH对镉胁迫大麦活性氧代谢的影响及基因型差异。结果表明,Cd处理大麦叶片中O₂^?和H₂O₂、MDA累积程度与其耐镉性相关,敏感基因型中积累更为严重。镉胁迫诱导萎缩不知尤其是根中的抗氧化酶活性的上升;东17地上部SOD、CAT和APX活性,在Cd处理中期较对照显著下降,APX同功酶分析显示,其中cAPX活性降低尤其严重。添加20 mg L^-1GSH对5μM Cd胁迫下大麦氧化损伤有明显的缓解效应,显著减少了O₂^?和H₂O₂、MDA的累积,并能诱导地上部SOD、CAT及APX活性增强,敏感基因型的CAT和APX活性在Cd+GSH处理下甚至显著高于对照。POD和CAT在表达水平对镉胁迫条件作出响应,但其与酶活性变化并不一致。更多还原

【Abstract】 Cadmium（Cd）is one of the most deleterious heavy metals to both plants and animals and has no beneficial biological function in the aquatic or terrestrial organism,but can be absorbed and accumulated easily by plants,while high accumulation of Cd in plants not only affects crop yield and quality badly,but also gives rise to a threat on human health via food chain.Although several approaches have been proposed to reduce soil Cd level. including the use of hyper-accumulating plants,none have been effectively applied. Considering large-scale medium/slightly contaminated farmlands such approaches as selection/breeding of crop genotypes/cultivars tolerant to Cd toxicity and with low Cd accumulation in edible parts,the improvement of agronomic practice and application of chemical regulators which can reduce plant Cd uptake would be a cost-effective and practical substitute mode to fully utilize natural resource and guarantee safe food production.Reduced glutathione（GSH）is the direct substrate for PCs synthesis,and the roles of GSH and PCs in heavy metal tolerance were reported by many researchers. Accordingly,the present study was carried out to select Cd- tolerant and sensitive genotypes,and to elucidate the mechanism of mitigative effect of GSH application on Cd toxicity and its genotypic difference by examining growth parameters,photosynthesis, anatomic structure,reactive oxygen species（ROS）metabolism and other physio-chemical responses of the two selected genotypes differing in Cd tolerance.The main results are as follows:1.Two successive hydroponic experments were carried out to identify barley varieties tolerant to Cd toxicity via examing SPAD value,plant height,dry weight,tillers per plant,root length and volume,and biomass accumulation.The results showed that SPAD value,plant height,dry weight,root length and volume,and biomass accumulation were significantly reduced in the plants grown in 20μM Cd compared with control,and Zn,Mn and Cu absorption and translocation were also strictly hindered.There was a highly significant difference in the decline of these growth parameters among genotypes.Weisuobuzhi and Jipi 1 showed the least reduction in both experiments,suggesting their high tolerance to Cd toxicity,while Dong 17 and Suyinmai 2 with the greatest decline and toxicity symptoms appeared rapidly and severely,donoting as Cd-sensitive genotypes.In addition,significant genotype difference in Cd concentration was also found,with Weisuobuzhi containing the highest,and Jipi 1 the lowest Cd concentration in shoots.2.Hydroponic experiments was undertaken,using two selected genotypes Weisuobuzhi （Cd-tolerant）and Dong 17（Cd-sensitive）exposed to 4 treatments of 0μM Cd（control）, 5μM Cd（Cd）,20 mg L^-1GSH（GSH）,and 5μM Cd+20 mg L^-1GSH（Cd+GSH）,to study the genotypic difference in the effect of exogenous GSH on barley growth, nutrient uptake and translocation under Cd stress.The results indicated that the tolerant genotype Weisuobuzhi,compared with the sensitive genotype Dong 17,showed much slighter visual leaf Cd toxic symptoms of necrotic patches but with significantly higher Cd concentration in shoots after 25 d 5μM Cd exposure.Cadmium stress seriously inhibited Zn,Mn and Cu absorption and translation in the both genotypes.Addition of 20 mg L^-1GSH（Cd+GSH）significantly reduced Cd uptake and accumulation compared with Cd treatment and being emphasized in Dong 17,but improved Zn,Mn and Cu uptake and its root/shoot concentration was even higher than control.Cadmium toxic symptoms was alleviated even more significantly in Dong 17 by GSH application, and the increases in plant height,root length and dry weight over its Cd treatment were much higher than that in Weisuobuzhi.3.The effect of exogenous GSH on barley cytoarchitecture and photosynthetic characteristics after 15 d Cd treatment and its genotypic differences were investigated. The results showed that 5μM Cd exposure caused a sharp decline（p≤0.05）in chlorophyll contents and net photosynthesis of the two barley genotypes with loosen chloroplast layer,and turgid grana and stroma lamellae in chloroplast were observed in both genotypes,granum stacking synchronously became incompact and some of them tend to degrade,furthermore,the deleterious effect of Cd was much severer in Cd-sensitive genotype Dong 17.Withal,increased accumulation in osmiophilic plastoglobuli and degradation of starch grain by Cd exposure were observed,and these deleterious effects were alleviated by GSH addition.Meanwhile,GSH supplement increased Pn,Tr and Sc of both genotypes under Cd stress.In addition,Cd stress impacted root ultrastructure,and resulted in ruleless sunken nuclear membrane,decline in the number and size of vacuole in root cells of both genotypes.Moreover,less number of cristae in mitochondria and plastids were found in the roots of Dong 17 under 5μM Cd,compared with control,while both of them remained unchanged and starch accumulated in plastids in Weisuobuzhi.Disservice in nucleus and mitochondria induced by Cd stress were almost recovered after GSH supplement.4.The effect of exogenous GSH on ROS metabolism in barley seedlings under Cd stress and genotypic differences was studied.The results showed that Cd stress induced the accumulation of O₂^?,H₂O₂ and MDA in leaves with genotypic difference,c.f.Dong 17 accumulated much more than Weisuobuzhi.The activities of antioxidant enzymes in Weisuobuzhi,particularly in roots,were significantly increased under Cd stress,while SOD,CAT and APX（especially cAPX）in the shoots of Dong 17 significantly decreased after 5-15 d Cd exposure.The accumulation of ROS and MDA was significantly reduced by GSH supplement.The induction of antioxidant enzymes such as SOD,CAT and APX activityies could be beneficial to decrease ROS and MDA accumulation to alleviate Cd induced oxidative stress.In Dong 17,CAT and APX activity were even higher than control after the addition of 20 mg L^-1GSH.POD and CAT responsed to Cd stress at expression level,but was not consistent with the trends of its activity.更多还原