【作者】 陈飞；

【导师】 邬飞波；

【作者基本信息】 浙江大学 ， 作物学， 2009， 博士

【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；对于大面积污染程度中、轻度的农田，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，而深入开展作物镉耐性和积累基凶型差异机理的研究是开展相关作物育种和生产调控的基础。本研究在筛选镉耐性与积累不同大麦基因型的基础上，研究其镉吸收和积累的规律及基因型差异的生理机理，鉴定耐镉／低积累相关的目标蛋白和相关基因，进一步从分子水平上探讨大麦镉吸收与转运机制，同时探讨了外源一氧化氮（NO）和还原型谷胱甘肽（GSH）对大麦镉吸收／转移与耐镉性影响的基因型差异及生理机制，为低镉积累育种与生产提供理论依据和技术指导。主要研究结果如下：1．大麦耐镉与籽粒低镉积累基因型筛选分析评估600份大麦基因型籽粒镉含量及其与Zn、Mn等微量元素积累的相关性，筛选籽粒镉低积累基因型；同时研究镉胁迫对大麦生长影响的基因型差异。结果表明，相同栽培条件下，600个大麦基因型籽粒镉含量变化范围为0-1．21 mg Cd kg^-1DW，基因型间差异显著，且有47．2％的参试大麦基因型籽粒镉含量超过WHO规定的最大允许值。连续二年的验证试验结果显示，籽粒镉含量基因型差异的变化趋势完全一致，其中Beitalys和上89-128籽粒镉含量最低，较籽粒镉高积累基因型鄂大麦6号和浙农8号极显著低97．5％。此外，600份基因型籽粒Zn、Mn、Fe和Cu等微量元素含量基因型间也存在显著差异，相关性分析结果显示，Mn与Cd存在显著正相关，而Cd和Zn、Cu、Fe的相关性不显著。以不同类型与生长习性大麦基因型为材料，两次连续的水培镉胁迫试验，结果表明，大麦幼苗对镉耐性和积累基因型间存在显著差异：筛选获得耐镉基因型萎缩不知和吉啤1号，镉敏感基因型东17和苏引麦2号。进一步分析测定植株不同器官中镉含量发现，四个基因型对镉的吸收和转移差异显著，其中耐性基因型萎缩不知镉含量最高，吉啤1号最低；而敏感基因型中则以东17地上部和地下部积累的镉含量较低。2．大麦籽粒成熟期镉的转运与积累及环境因子的影响采用离体穗培养方法，研究大麦籽粒成熟期镉积累动态、再转移及分配规律及环境因素对籽粒镉积累的影响。结果表明，离体穗不同器官中镉含量都随培养液中镉处理水平提高而增加。三个镉水平下，处理15 d后离体穗各部位镉含量依次都为：芒＞茎秆＞籽粒＞穗轴＞颖壳；镉积累量以籽粒中占的比例最大，占各部份总积累的51．0％。镉积祟动态变化结果显示，籽粒镉积累随处理时间的延长而上升：离体穗其他各部位锅积累处理1-5 d显著上升，5-10 d时则急剧下降，而处理12．5 d后各部位镉积累量又显著回升。此外，去芒和茎环割显著降低离体穗籽粒镉含量，其中以去芒处理尤为显著；培养液中添加蔗糖和Zn及提高环境湿度均显著降低镉向发育籽粒中的转移。这些结果表明，芒、穗轴和颖壳可能参与了镉向籽粒的再转移，大麦成熟期镉的再分配也是影响最后籽粒质量的重要生理过程，提高环境湿度至RH90％或在培养液中加Zn，都能显著降低灌浆期大麦籽粒镉积累。3．大麦籽粒成熟期镉转运与积累的基因型差异以筛选获得的籽粒镉低／高积累基因型W6nk2／浙农8号为材料，离体穗培法研究木质部和韧皮部运输对大麦籽粒成熟期镉转移、分配的影响及基因型差异。结果表明，0．5μM Cd处理，W6nk2离体穗对镉的吸收能力显著低于浙农8号，而随着镉处理浓度的增加，W6nk2离体穗籽粒镉含量也大幅上升，培养液镉水平增至5μM Cd时，与浙农8号的籽粒镉含量无显著差异。提高环境湿度和去芒均显著抑制镉向浙农8号和W6nk2离体穗各部位的转移，尤其以浙农8号籽粒镉含量下降更为显著。低浓度镉处理下茎环割对两个基因型籽粒镉含量影响不显著，而高镉浓度处理下茎环割显著降低了两个基因型籽粒镉含量，其中对W6nk2影响尤为显著。这些结果表明，不同的木质部和韧皮部运输能力是导致两个大麦基因犁籽粒镉积累差异的主要原因，通过选育木质部运输能力较低的大麦品种，同时在籽粒成熟期提高环境湿度或者调控韧皮部运输均有利于降低籽粒镉的积累。4．大麦耐镉与低镉积累相关蛋白与基因特异表达分析利用基因芯片技术，分析了耐镉性和籽粒镉积累显著差异的大麦基因型幼苗在镉胁迫下转录组的变化，结果显示，镉胁迫分别诱导四个大麦基因型叶片转录水平发生变化，不同基因型间差异显著。其中籽粒镉低积累基因型W6nk2和敏感基因型东17中差异表达的基因数分别高于籽粒镉高积累基因型浙农8号和耐镉基因型萎缩不知。分别对两组基因型镉胁迫下差异表达基因比对发现，5μM Cd处理诱导W6nk2大量的运输载体相关基因上调表达，如ABC转运蛋白、ATPase、以及Zn、Fe离子运输载体等基因，说明W6nk2籽粒镉低积累特性可能与这些转运载体基因的上调表达相关。镉胁迫引起东17中大量光台作用、运输、信号转导等相关基因显著下调，特别是PC合成酶基因（PCS）表达的下调；但萎缩不知中并未检测到PCS的变化，同时部分抗氧化酶（如CAT等）基因上调表达，说明植物体内PCs的合成及抗氧化能力的提高对增强耐镉性具有重要的作用。蛋白双向电泳结果显示，镉胁迫引起不同耐镉性大麦幼苗蛋白质组水平发生变化，5μM Cd处理后两个基因型中共检测到112个差异表达的蛋白点。进一步通过质谱分析发现，镉胁迫诱导部分抗氧化酶、信号转导、运输以及光合和碳水化合物代谢等蛋白质表达发生变化，特别是H⁺-转运二区室ATP酶和甘氨酸脱羧酶亚基在萎缩不知中表达上调，而在东17中保持不变，说明两者可能与大麦耐镉性紧密相关。5．外源NO和GSH对镉胁迫下大麦生长及微量元素吸收的影响及基因型差异以耐镉基因型萎缩不知和镉敏感基因型东17为材料，水培试验，研究外源NO（SNP）、GSH对镉吸收转移及耐镉性影响的基因型差异及生理机制。结果表明，5μMCd处理下，添加20 mg L^-1 GSH均显著降低了大麦幼苗对镉的吸收和积累；两个基因型地上部和地下部镉含量在添加0．25 mM SNP后也显著下降，但地下部镉累积量却出现不同程度的提高，以敏感基因型东17根部镉积累量提高最为明显。NO和GSH均显著缓解大麦幼苗镉毒害症状，两个基因型株高、根长及生物量都比Cd处理显著提高，尤其对敏感基因型Dong17缓解效应更佳。表明GSH主要通过减少植物镉吸收和积累缓解镉毒害，而NO则可能存在其他缓解机制。6．外源一氧化氮对大麦镉毒害缓解效应及基因型差异研究了外源NO对镉胁迫大麦光合作用、细胞超微结构及活性氧代谢的影响及基因型差异。结果表明，添加0．25 mM SNP能显著缓解镉胁迫引起的叶绿体和根系细胞结构的损伤，基本恢复叶绿体片层结构的损伤，嗜锇颗粒的数量显著减少，淀粉粒积累增加：有效提高了根系分生组织细胞核膜的稳定性和完整性。外源SNP对镉胁迫引起的大麦氧化损伤有明显的缓解效应，显著减少了叶片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+NO处理下甚至显著高于对照。对基因表达水平分析结果显示，cAPX在Cd+NO处理后表达量显著回升，与其酶活测定结果一致，而POD和CAT1在表达水平与其酶活性变化并不一致。说明NO通过维持镉胁迫大麦叶绿体和根系细胞结构的稳定性并诱导抗氧化酶活性，有效预防和清除镉胁迫引起ROS的积累，维持体内氧化还原平衡与较高的光合速率，从而起到解毒作用。此外，镉胁迫诱导大麦体内NO激发，5μMCd处理1d后，萎缩不知及东17叶片NO含量迅速上升，但东17根系NO含量未发生变化：两个基因型叶片NOS活性同样受镉诱导应激上升，而NR活性仅在萎缩不知叶片中上升，东17中则下降。说明镉胁迫诱导NOS和NR活性的上升，增加NO合成，对提高大麦耐镉性具有重要作用。更多还原

【Abstract】 Cadmium （Cd） is one of the most deleterious heavy metals to both plants and animalsand has no beneficial biological function in the aquatic or terrestrial organism, but can beabsorbed and accumulated easily by plants, while high accumulation of Cd in plants notonly affects crop yield and quality badly, but also gives rise to a threat on human health viafood 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 asselection/breeding of crop genotypes/cultivars tolerant to Cd toxicity and with low Cdaccumulation in edible parts, the improvement of agronomic practice and application ofchemical regulators which can reduce plant Cd uptake would be a cost-effective andpractical substitute mode to fully utilize natural resource and guarantee safe foodproduction. Accordingly, the present study was carried out to elucidate the physiologicaland molecular mechanism of Cd uptake and translocation in barley, and to identify specificproteins and relevant genes for Cd- tolerance/low accumulation through proteome andgenome analysis, based on the selection of genotypes differing in Cd accumulation andtolerance. Meanwhile, we investigated the possibility to reduce Cd uptake andaccumulation in barley plants by application of exogenous nitric oxide （NO） andglutathione （GSH） and the protective effect of exogenous NO against Cd-induced growthinhibition, oxidative stress, and damage in ultrastructure and photosynthesis in the twoselected genotypes differing in Cd tolerance. The main results were summarized follows:1. Identification of barley genotypes with low grain Cd accumulation and tolerance toCd toxicity.The variation in grain Cd concentrations was evaluated among 600 barley genotypesgrown in the same field condition to select low Cd accumulating genotypes. The resultsshowed that there is considerable genotypic variation in grain Cd concentrations in barleygrain samples, with the mean concentration of 0.16 mg kg^-1 DW and the variation of 0 （notdetected） to 1.21 mg kg^-1 DW, and 47.2% of the grain samples exceeded the maximumpermissible concentration （MPC） for Cd in cereal grains. In addition, differences betweengenotypes over the two years were fairly consistent, and Beitalys and Shang 98-128 showedthe lowest grain Cd concentration, being 97.5% lower than that in the two highest Cdaccumulators E-barley 6 and Zhenong 8 in the second harvest year. The great genotypic differences in Cd concentrations indicated that it is possible to lower Cd content of barleythrough cultivar selection and breeding for use at sites where Cd concentration in grainexceeds the MPC. Significant genotypic difference was also found in microelementconcentrations. Correlation analysis showed that only Mn accumulation is synergetic withCd accumulation, despite slightly positive relationship between Cd and Zn, Cu, or Fe inaccumulation in barley grains.Two successive hydroponic experments were carried out to identify barley varietiestolerant to Cd toxicity via examing SPAD value, dry weight, plant height, root length andvolume, tillers per plant, and biomass accumulation. The results showed that SPAD value,dry weight, plant height, root length and volume, and biomass accumulation weresignificantly reduced in the plants grown in 20μM Cd compared with control. There was ahighly significant difference in the decline of these growth parameters among genotypes.Weisuobuzhi and Jipi 1 showed the least reduction in both experiments, suggesting theirhigh tolerance to Cd toxicity, while Dong 17 and Suyinmai 2 with the greatest decline andtoxicity symptoms appeared rapidly and severely, donoting as Cd-sensitive genotypes. Inaddition, significant genotype difference in Cd concentration was also found, withWeisuobuzhi containing the highest, and Dong 17 had lower Cd concentration comparedwith Suyinmai 2.2. Cadmium transiocation and accumulation in developing barley grains and asaffected by some environmental factors.In order to study the transport of Cd into the developing grains, detached ears oftwo-rowed barley cv. ZAU 3 were cultured in Cd stressed nutrient solution containing themarkers for phloem （rubidium） and xylem （strontium） transport. Cd concentration in eachpart of detached spikes increased with external Cd levels, and Cd concentration in variousorgans over the three Cd levels of 0.5, 2, 8 M Cd on 15-day Cd exposure was in the order:awn > stem > grain > rachis > glume, while the majority of Cd was accumulated in grainswith the proportion of 51.0% relative to the total Cd amount in the five parts of detachedspikes. Cd accumulation in grains increased not only with external Cd levels but the time ofexposure contrast to stem, awn, rachis and glume. Those four parts of detached spikeshowed increase Cd accumulation for 5 days, followed by sharp decrease till day 10 andincrease again after 12.5 days. Awn-removal and stem-girdling markedly decreased Cdconcentration in grains, and sucrose or zinc （Zn） addition to the medium and higher relativehumidity （RH） also induced dramatic reduction in Cd transport to developing grains. Theresults indicated that awn, rachis and glume may involve in Cd transport into developing grains, and suggested that Cd redistribution in maturing cereals be considered as animportant physiological process influencing the quality of harvested grains. Our resultssuggested that increasing RH to 90% and Zn addition in the medium at grain filling stagewould be beneficial to decrease Cd accumulation in grains.3. Genotypic difference of Cd transporting into the developing grains.Genotypic difference of Cd transport into the developing grains was investigated usingdetached ears of two barley genotypes cultured in nutrient solution containing 0.5 and 5μMCd. The results showed that Cd concentration in each part of ears in W6nk2 （low grain Cdaccumulation genotype） was extremely lower than these in Zhenong 8 （high accumulator）in 0.5μM Cd treatment. However, grain Cd concentration of W6nk2 increased obviouslywith external Cd levels, and even reached the level of Zhenong 8 in 5μM Cd treatment.Awn-removal, high relative humidity （RH） and sucrose addition markedly decreased Cdconcentration in grains of Zhenong 8. but affected slighter in Cd transport to the grains ofW6nk2. Stem girdling also reduced the Cd transport to developing grains in 5μM Cdtreatment, especially for the low kernel Cd accumulation genotype W6nk2, while no effecton Cd transport to developing grains of both genotypes in low Cd treatment. Our resultssuggested that the different ability of Cd translocation in the xylem and phloem sap mightcontribute to the difference in grain Cd accumulation of the two genotypes. The results alsosuggest that screening the cultivar with low xylem transport, and regulated the phloemtransport at grain filling stage would be beneficial to decrease Cd accumulation in grains.4. Identification of specific proteins and relevant genes for Cd- tolerance/lowaccumulation.To begin to unravel the as-yet poorly understood molecular mechanisms of Cd uptake,translocation and tolerance in barley plants, we compared gene expression in four barleygenotypes differing in Cd tolerance and accumulation using cDNA microarrays. BarleylAffymetrix GeneChip was used to analyze the transcriptional response of barley plantsexposure to 0 （control） and 5μM Cd. The results showed that there was significantdifference on expression response of genes related to Cd stress among four genotypes.Transcriptional levels of 514 and 658 genes in low Cd accumulation genotype W6nk2 andCd sensitive genotype Dong 17 altered after plants exposed to 5μM Cd for 15 d, while only413 and 305 genes altered in Zhenong 8 （high Cd accumulation genotype） and Weisuobuzhi（Cd tolerant genotype） under Cd stress. The up-regulated transcript levels of genes relatingto metal transporters in plants were observed, such as ABC transporter, P-type ATPase, iron-phytosiderophore transporter and zinc transporter protein ZIP1 in leaves of W6nk2after Cd exposure, which might contribute to its low Cd accumulation characterization.Compared to Cd tolerant genotype Weisuobuzhi, more metal transport, photosynthesis andsignal transduction related genes were detected in Cd sensitive genotype Dong 17 under Cdstress. Meanwhile, the transcript level of PCS was not changed in Weisuobuzhi butdown-regulated in Dong 17, in addition, some antioxidant enzymes （eg. CAT） wereup-regulated in Weisuobuzhi, indicating that increasing synthesize of PCs and the activityof antioxidant enzymes were important to Cd tolerance in barley plants. In the other hand,proteomic analysis was used to investigate the Cd stress-responsive proteins in differentbarley genotypes, significantly genotypic difference on expression response of proteinsrelated to Cd stress was also found in two barley genotypes. Mass spectrometry analysisand database searching helped us to identify 28 spots representing 17 different proteins,involving in regulation of antioxidant enzymes, signal transduction, metal transport andphotosynthesis. Two of them (c.f. H^?-transporting two-sector ATPase and putative glycinedecarboxylase subunit) were up-regulated in Weisuobuzhi but not changed in Dong 17,indicating that these two proteins might play an important role in Cd tolerance.5. Effect of external NO and GSH supply on plant growth and microelementsaccumulation of two barley genotypes under Cd stress.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）, 5μM Cd+0.25mM SNP （Cd+NO）, and 5μM Cd+20 mg L^-1 GSH （Cd+GSH）,to study the genotypic difference in the effect of exogenous NO and GSH on barleygrowth, Cd and other nutrients uptake and translocation under Cd stress. The resultsindicated that addition of 20mgL^-1 GSH （Cd+GSH） significantly reduced Cd uptake andaccumulation compared with Cd treatment and being emphasized in Dong 17. While0.25mM SNP added （Cd+NO） only reduced the concentration of Cd, but increased theaccumulation in roots of both genotypes, especially in Dong 17. Cadmium toxic symptomswas alleviated even more significantly in Dong 17 by NO and GSH application, and theincreases in plant height, root length and dry weight over its Cd treatment were muchhigher than that in Weisuobuzhi. It could be concluded that reduced Cd concentration andaccumulation in plants could be one of principal protective mechanism for the exogenousGSH in cytoprotection against Cd toxicity. GSH would be suitable for the edible cropsgrown in Cd soils to reduce Cd accumulation and alleviate phyto-toxicity of Cd. 6. Mechanism of alleviation effects of NO application on Cd toxicity and its genotypicdifference.A greenhouse hydroponic experiment was carried out, using two barley genotypesdiffering in Cd tolerance, to evaluate the protective effect of exogenous NO againstCd-induced oxidative stress, and ultrastructure and photosynthesis damage. Addition of0.25 mM SNP in 5μM Cd culture medium significantly alleviated Cd-induced growthinhibition, and its beneficial effect was observed in all growth parameters especially for thesensitive genotype Dong 17. Exogenous NO dramatically depressed ROS and MDAaccumulation, compared with 5μM Cd treatment, meliorated Cd-induced damages on leafand root ultrastructure, and increased chlorophyll content, Pn, Gs and Tr, thus improvedphotosynthesis efficiency. Meanwhile, exogenous NO counteracted Cd-induced time- andgenotype- dependent response of antioxidant enzymes, via suppressing the Cd-induceddramatic increase of POD activities in shoots of both genotypes recovery to near the controlvalues, and by elevating depressed APX, and CAT activities in Dong 17 after 10, and 15 dtreatment. The examination of APX and SOD isoenzymes in leaves revealed NOsignificantly increased sAPX and MnSOD activities in the both genotypes, and stronglystimulated Cd-induced decrease in cAPX （the major isoenzyme in barley leaves） insensitive genotype, but down-regulated the increased level in Weisuobuzhi. The results ofRT-PCR showed that POD, CAT1 and cAPX responded to Cd stress at transcript level.External NO up-regulated root and leaf cAPX and leaf CAT1 expression in Dong 17 toachieve stimulation. It could be concluded that improved photosynthesis efficiency and themembrane-stabilizing/integrity effect could be principal protective mechanism for theexogenous NO in cytoprotection against Cd toxicity. The results also suggested a practicalpotential for NO as a potent antioxidant in plants and that its action may, at least in part, beexplained by its ability to directly and indirectly scavenge ROS.更多还原