【作者】 李丰涛；

【导师】 祁建民；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2013， 博士

【摘要】 随着化工、采矿行业的加速发展及农业中大量化肥和农药使用,重金属不可避免地被带入到土壤环境中,破坏了土壤生态环境。农作物吸收重金属进入食物链,进而危害人体健康甚至造成生命威胁。植物修复技术是一种利用植物吸收带走土壤中重金属的技术,具有环保洁净、操作简便、成本低廉、不会造成二次污染等优良特征。红麻(Hibiscus cannabinusl)是锦葵科木槿属一年生韧皮纤维作物,又是一种能源植物,不进入食物链,具有耐旱、耐盐碱、速生、生物产量高、根系发达、抗逆性强等特征。我国红麻种质资源库大约有1700多份种质资源,为筛选可用于重金属植物修复的品种奠定了良好的基础。筛选高耐性、高富集重金属且高产优质的品种,不仅对土壤重金属污染修复有重要意义,而且可以带来一定的经济价值。本研究主要做了以下工作,对福建闽中南农田土壤环境做了调查和测定,并对种植红麻体内重金属含量进行了测评,研究了红麻对重金属的吸收特征;采用水培实验的方法研究了重金属镉对红麻幼苗生长的影响及红麻镉吸收特征,筛选出了高耐、高富集镉的红麻品种；以水培实验筛选出的四种不同类型的红麻品种为材料,进行盆栽土培实验,进一步研究了红麻对重金属镉的吸收累积特征及其品种间的差异；研究了高耐品种和低耐品种的抗氧化系统对镉胁迫响应的差异；研究镉胁迫对红麻根尖细胞结构的影响；采用蛋白组学的方法研究了镉胁迫下红麻叶片的蛋白组响应及还原型谷胱甘肽对镉胁迫的缓解作用。主要研究结果如下：1.闽中南红麻种植田土壤重金属含量及其富集特征的研究通过野外采样和室内分析相结合的方法,分析了闽中南4个地点山地农田土壤重金属全量和有效态含量,研究了农田内12个采样点的种植红麻福红952对Cu、Cd、Zn、Cr、Ni和Pb6种重金属的吸收与富集特征。结果表明,漳浦县前亭镇某村山地Cr和Ni平均含量达187.1mg·kg-1和109.5mg·kg-1,分别是土壤环境质量标准(Ⅱ级)的1.3倍和2.7倍,是福建省土壤元素背景值的4.5倍和7.6倍,是全国土壤元素背景值的2.9倍和4.8倍。红麻对重金属的迁移能力较强,当季对Cu最高累积量为251.6g·hm-2;Pb最高累积量为263.4g·hm-2;Zn最高累积量为1021.1g·hm-2;Cr最高累积量为105.0g.hm-2;Cd最高累积量为8.6g·hm-2;Ni最高累积量为112.1g·hm-2;。2.不同红麻品种苗期对镉胁迫的适应性、吸收特征及耐性评价以53个产自不同地域和表型特征的红麻品种为材料,通过设置Cd浓度为30μM进行水培实验,结果表明Cd胁迫对红麻株高、根长、色素含量、地上部和根系生物量等都有显著影响,且浓度越高对红麻生长影响越大。采用隶属函数对红麻耐性指标进行聚类分析,将红麻品种分为3类：高耐型、中耐型、低耐型。同时,测定了红麻苗期Cd吸收特征和富集量,并对耐性指标和Cd累积总量进一步聚类分析,将供试红麻品种分为4类：高耐高吸收型、高耐低吸收型、低耐低吸收型和低耐高吸收型。3.不同基因型红麻品种镉吸收的富集特征以四种不同Cd吸收类型中的代表品种为材料,即高耐高吸收型(福红991)、高耐低吸收型(粤743)、低耐低吸收型(ZM412)和低耐高吸收型(福红航992),研究了不同基因型红麻品种镉吸收的富集特征。红麻品种各器官中镉含量随镉浓度增加呈上升趋势,在高耐镉品种各器官中的镉含量顺序：根>茎>叶>麻皮,低耐镉品种各器官中的镉含量顺序：根>叶>茎>麻皮。非施肥和施肥条件下,红麻品种间地上部Cd含量存在着显著差异。当Cd处理浓度为120mg/kg时,在施肥条件下,福红991和福红航992地上部Cd含量均超过100mg/kg,其中福红991达到了117.96mg/kg。在Cd污染浓度为10mg/kg土壤中,如果种植福红航992每年每公顷可以产原麻3662.8kg,同时清除土壤中Cd0.74kg/hm2;在土壤Cd浓度为50mg/kg中,如果种植福红991每年每公顷可以产原麻1400kg以上,同时可以吸附Cd2.58kg/hm2。4.红麻抗氧化系统对镉胁迫的响应水培实验结果表明,Cd胁迫处理抑制了红麻的生长,福红991和ZM412的抗氧化系统对Cd胁迫处理响应存在显著差异性。SOD、POD和CAT活性在Cd处理梯度上,两个品种均有不同程度的波动,不过福红991的GR活性,在Cd浓度为20μM时达到最大值,随Cd处理浓度不断升高而降低,但仍高于对照,说明了GR在抵御Cd胁迫中的重要性,同时暗示了在不同Cd胁迫水平上,福红991保持了较好的细胞膜稳定性。为了监测Cd污染对红麻生长的影响,福红991的GR活性可以作为生物标记,此外,ZM412的MDA含量可作为生物标记。5.不同镉处理浓度对红麻根尖细胞结构的影响Cd胁迫处理,导致了红麻根系活力降低和根膜透性增加,说明Cd对红麻根系造成了严重损伤。当Cd浓度较低时,细胞壁和细胞膜出现黑色沉积,将Cd离子挡在原生质之外,起到保护作用,但随着Cd处理浓度的增加,红麻根尖皮层细胞出现质壁分离和原生质膜破裂现象,且细胞内细胞器逐渐减少,核膜和核仁解体,游离在细胞质中,这些超微结构的改变给红麻细胞造成了不可拟的损伤。6.添加不同浓度还原型谷胱甘肽对红麻镉毒害的缓解效应添加不同浓度GSH,对提高两个基因型红麻幼苗的根长、株高、地上部鲜重、干重及叶绿素含量均有显著效果,在GSH处理浓度为50μM时,两品种的根长与对照之间无明显差异,福红991株高、地上部鲜重和干重与对照无显著差异,当GSH浓度为150μM时,叶片的叶绿素含量基本恢复到对照水平,说明GSH起到了缓解Cd对红麻的毒害作用。对红麻生长相关指标进行综合分析,当GSH浓度为50gM时,GSH对红麻缓解效应达到了一个比较理想阈值,在浓度为100gM时有小幅下降,浓度更高时与50μM相差不大,考虑到GSH应用到环境中治理镉毒害的实用性及价格因素,认为50μM GSH对红麻的镉毒害缓解作用最佳。7.红麻叶片蛋白质组对中低浓度镉毒响应的基因型差异及外源谷胱甘肽的缓解效应本节研究了50μM GSH对Cd胁迫下红麻叶片蛋白质谱表达的影响及基因型差异。结果表明,在Cd胁迫下ZM412的生长受到更加明显的抑制。经软件分析,鉴定了存在显著差异表达的蛋白点72个,LC-MS/MS质谱分析鉴定出50个差异蛋白点。其中有8个蛋白,在Cd胁迫时上调表达,应该与Cd耐性相关,它们是参与光合作用、防御和运输的蛋白。另外,GSH缓解镉胁迫响应蛋白有21个,这些蛋白是参与光合作用、蛋白质合成、信号转导、防御及能量和物质代谢的蛋白,这些结果可能暗示了,GSH在这些方面的作用提高了红麻耐镉能力。更多还原

【Abstract】 With the accelerated development of the chemical industry, mining industry and large amounts of chemical fertilizers and pesticides used in agriculture, heavy metals were inevitably brought into the soil with destroying the ecological environment. The crops absorbed heavy metals into the food chain, thereby endangered human health and even life-threatening. Phytoremediation technology was a method of using plants to clear heavy metals from polluted soil, with excellent characteristics of environmental protection, convenient operation, low cost and no secondary pollution.Kenaf (Hibiscus:Malvaceae) was an annual bast fiber crop, and it was also an bast fiber plant not entering the food chain Which was drought tolerance, alkali resistant, fast-growing, high productive, rich root, anti-stress. The genetic resources center of kenaf had about over1700germplasm resources, which laid a good foundation for selecting materials of using for phytoremediation. Therefore, selecting materials of high endurance, enrichment and yield was significant to remedy heavy metals in contaminated soil. At the same time, it would brought some economic value.In this study, we investigated heavy metal concentrations and bioaccumulation of kenaf growed in south-central farmland of Fujian province. We studied the effects of cadmium on the growth and absorption characteristics of kenaf seedlings by doing hydroponic experiments, and selected varieties which had high resistance and enrichment to cadmium. We did pot culture experiments using the four different varieties which were selected from the hydroponic experiments, studying the characteristics of the accumulation of cadmium between the varieties. Antioxidant system differences in response to cadmium stress were studied between the high resistant varieties and low resistant varieties. By using the proteomics, that responsed to cadmium stress of kenaf leaves and mitigating effect of GSH were studied.1. Heavy metal concentrations and bioaccumulation of kenaf growed in south-central farmland of Fujian province.By the means of field investigation and laboratory analysis, total contents and Bio-available contents of soil heavy metals were analyzed by collecting samples from cultivated soil in four areas of the center and south of Fujian Province, and uptake and accumulation of Cu, Cd, Zn, Cr, Ni and Pb in12sample sites of Fuhong952were also analyzed. The results showed that Cr(187.1mg-kg"1) and Ni(109.5mg·kg-1) in cultivated soil of Zhangpu far exceeded soil environmental quality standards (grade II), were respectively4.5times and7.6times background value of soil elements in Fujian Province, and is respectively2.9times and4.8times of the background value of soil elements in China Though content of Cd was low in four areas, Bio-available contents accounted for more than15%of total content. The bioaccumulation factor of Cd in kenaf were more than1. The highest accumulation contents of shoots.2. Adaptability, accumulation and tolerance evaluation in Cd-stressed kenaf cultivarsCollected53kenaf varieties from different regions and phenotypic characteristics, the concentration of Cd was30μM in hydroponics experiments. The results showed that Cd stress had significant effects on height, root length, pigment, shoot and root biomass of kenaf plants. The higher the concentration, the greater the impact on kenaf growth. The tolerance index was analyzed in clustering based on the membership function, kenaf varieties were divided into three kinds:high-tolerance, middle-tolerance, low-tolerance. In addition, we measured uptake and accumulation of Cd in seedling stage. The tolerance index and total accumulation were analyzed in clustering. Kenaf varieties were further divided into four kinds:high-tolerance and high-accumulation, high-tolerance and low-accumulation, low-tolerance and high-accumulation, low-tolerance and low-accumulation.3. Accumulation of Cd in different kenaf varietiesUsed representative varieties in four types [high-tolerance and high-accumulation (Fuhong991), high-tolerance and low-accumulation(Yue743), low-tolerance and high-accumulation(Fuhonghang992), low-tolerance and low-accumulation(ZM412)] like chapter2as materials, cadmium accumulation kept upward trend in various organs of kenaf with increasing concentration of cadmium, cadmium accumulation in various organs of the high-tolerance:root> stem> leaves> bark, cadmium accumulation in various organs of the low-tolerance:root> leaf> stem>bark. Under unfertilization and fertilization conditions, there were significant differences among shoots accumulation of kenaf varieties, Cd accumulation in shoot of Fuhong991and Fuhonghang992were more than100mg/kg as Cd concentration was120mg/kg, and Fuhong991was up to117.96mg/kg. Fuhonghang992per hectare per year could harvest raw fiber3662.8kg in the soil (Cd,10mg/kg), and remove Cd0.74mg/hm2from polluted soil. Fuhong991per hectare per year can produce raw fiber more than1400kg in the soil (Cd,50mg/kg) and uptake Cd2.58kg/hm2.4. Antioxidant system in response to cadmium stress of KenafAntioxidative enzymes and lipid peroxidation were analysed under control (0.5-strength Hoagland’s nutrient solution) or five levels of cadmium stress (0.5-strength Hoagland’s nutrient solution containing different concentrations of Cd2+) in two kenaf plants (FuHong991and ZM412), the results showed that cadmium stress effected the growth of Kenaf. In different Cd of enzyme(SOD, POD, CAT) activities, there were fluctuation in different degrees of two varieties FuHong991is more tolerant to cadmium than ZM412. The lipid peroxidation was enhanced only in the leaves of Cd-stressed ZM412. These findings indicate that GR might play important roles in Cd-stressed FuHong991and ZM412and that the leaf and root cell membranes of FuHong991have a greater stability than those of ZM412. For the purposes of pollution monitoring of Cd, the GR activity in the roots and leaves may serve as a biomarker of FuHong991, and MDA might serve as biomarker of ZM412.5. Effects Of cadmium stress on kenaf apical cell structureThis study carried out experiments to investigate changes of shape, inter structures and cell microstructures of kenaf roots under Cd stress using obersvation and biomicroscopic technique. With the increase of Cd concentration, in cells of root cortex plasmolysis and the destruction of plasmalemma became more serious, cell organelles disintegrated completely, nucleus membrane finally disappeared leading to nucleolus and nucleoplasm distributing in cytoplasm, dark sediment distributed in cytoplasm, plasmalemma and vessel walls.6. Mitigating effection of cadmium toxicity of Kenaf by adding different concentrations of GSHBy adding different concentrations of GSH, there were significant effects to increase the root length, plant height, shoot fresh weight, dry weight and chlorophyll content of Kenaf seedlings of two varieties. When concentration of GSH was50μM, there was no significant difference in root length between two varieties and the control. There was no difference in plant height, shoot fresh weight, dry weight between Fuhong991and the control. When the concentration of GSH was150μM, the chlorophyll content of leaves basically recovered to the control levels, which showed that GSH played alleviate toxic effect of Cd on Kenaf. When concentration of GSH was50μM, alleviating effect of GSH reached a more ideal threshold. Comprehensive comparative analysis of results showed concentration of50μM could serve as an effective mitigation of Cd.7. The proteomic of Kenaf leaves of different genotypic responsed to Cd stress and alleviating effect of GSHBy using GSH (50μM), the protein expression map and genotypic difference of Cd stress of Kenaf leaves were studied. The results show that the growth of ZM412was significantly inhibited by Cd stress. By software analysis,72protein spots that existed differential expression were identified. We identified50proteins by LC-MS/MS mass spectrometry analysis.8proteins upregulated the expression under Cd stress. Which should be associated with Cd tolerance, and they were involved in photosynthesis, defense and transport. In addition,21proteins responsed to alleviating Cd stress of GSH, these proteins were involved in photosynthesis, protein synthesis, signal transduction, defense, energy and material metabolism. It suggested that GSH played important role in improving tolerance of Kenaf Cd stress.更多还原