【作者】 佘玮；

【导师】 揭雨成；

【作者基本信息】 湖南农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 采矿业、工业生产和高强度的农业活动将大量的重金属带入土壤环境,破坏土壤生态系统。重金属通过植物吸收进入食物链,进而在人体内长期累积,严重危害人们的健康。植物修复技术以其操作简便、环境友好、修复成本低廉性和工程处理简易等优点日渐受到人们重视。苎麻又名“中国草”,具有抗逆能力强、繁殖能力强、根系庞大、生物产量高等特点。我国苎麻种质资源十分丰富,为筛选可应用于重金属植物修复的品种提供了有利的条件。选择重金属耐性强、富集量大且还能高产优质的品种对修复重金属污染土壤具有实际意义。苎麻主要作为纺织等工业原料,通过食物链危害人体健康的风险较小,在治理土壤污染的同时还有一定的经济收益。本文首先采用定位试验研究了矿区野生型苎麻对重金属的吸收积累特征；然后采用盆栽试验和大田微区试验相结合的方法研究了重金属镉对苎麻栽培品种生长的影响、筛选耐镉品种的方法和指标、研究了苎麻对重金属镉的吸收积累特征及其品种间差异；最后采用Solexa技术研究苎麻镉胁迫响应基因表达。主要研究结果如下：1、矿区野生型苎麻对重金属吸收和积累特征石门雄黄矿区As污染严重,伴随Cd、Sb污染和轻微的Pb污染；冷水江锑矿区Sb为主要污染物,伴随Cd、As、Pb污染；浏阳七宝山矿区Cd污染严重,伴随Pb、Zn、Cu污染。Sb和As在苎麻不同部位间的分布次序为叶片中含量最高,其余重金属在部位间分布没有规律。所有采样点苎麻地上部的Cd含量比一般植物的Cd含量大2～10倍,As含量大9.9～147.5倍,Sb含量大1.2～338.4倍。苎麻地上部Cd、As含量与土壤中Cd、As总量显著相关,苎麻地上部Pb、Sb、Zn和Cu含量与土壤Pb、Sb、Zn和Cu总量没有显著相关性。Cd富集系数和转移系数最高值为2.07和3：As富集系数和转移系数最高值为1.04和12.42,Sb富集系数和转移系数最高值为1.91和9.04。苎麻生物量大,地上部Cd、Pb、As、Sb、Zn和Cu的积累量分别高达0.11、1.17、0.72、7.97、6.71和1.69 kg／hm2。说明苎麻适合用作矿区复合重金属污染土壤的环境治理和修复。2、苎麻栽培品种对镉适应性和耐性评价盆栽试验和大田微区试验结果表明Cd对苎麻株高、茎粗、皮厚、生物量、叶绿素含量等有显著影响,且浓度越高,影响程度越大。但盆栽试验中,石阡竹根麻、宜春红心麻和川苎1号三个品种在Cd浓度23～46mg／L下的地上部生物量均高于对照；大田微区试验中,石阡竹根麻和川苎1号在25mg／kgCd处理下的生物量较对照分别增加了38%和36%。通过对苎麻耐性指标综合评价,发现盆栽试验和大田微区试验结果一致,建立了以珍珠岩为基质的营养液盆栽试验筛选苎麻耐镉品种的方法和指标。通过聚类分析将供试品种分为3类：高耐型,中耐型和低耐型。3、苎麻栽培品种镉吸收积累特征盆栽试验结果表明苎麻各器官Cd平均含量按大小排序依次为茎皮＞根＞骨＞叶,苎麻地上部Cd含量与土壤Cd浓度呈显著正相关(0.963)。盆栽试验中Cd处理为23、46、91和182mg/L时,苎麻地上部Cd平均含量分别为22.93,37.22,71.27和88.08mg／kg。大田微区试验结果表明茎皮＞麻骨＞叶,茎皮中Cd含量显著高于其它器官。大田微区试验对照(1.65mg／kg、Cd处理25mg／kg和100mg／kg时苎麻地上部Cd平均含量分别为7.75,28.19和45.11 mg／kg。苎麻地上部Cd积累量品种间差异显著,中苎1号和富顺青麻Cd积累量较强,土壤Cd浓度为1.65mg／kg时,种植中苎1号全年每公顷可产原麻2300kg的同时能从土壤中带走Cd 0.25kg／hm2,土壤Cd浓度为25mg／kg和100mg／kg时,种植富顺青麻全年每公顷可产原麻1900kg以上,同时可分别吸附Cd0.76和0.97kg／hm2。以耐性隶属函数值和地上部Cd含量为指标将苎麻品种聚类为4个不同类型,高耐低吸收型、高耐高吸收型、低耐低吸收型和低耐高吸收型。4、苎麻镉胁迫响应基因表达研究构建了对照(未加Cd处理)和100mg／kgCd处理的苎麻根系数字表达基因数据库。通过Solexa测序分别获得了3,654,395和3,572,333个标签(tag,Clean tag分别占测序总数的93.18%和93.76%。通过数据分析,在对照和处理间共筛选到差异表达基因3887个,包含2883个上调基因和1004个下调基因,blast比对发现其中大部分差异表达基因为未知基因,有待进一步发掘利用。对功能已知差异表达基因进行功能注释富集分析,在分子功能上这些基因主要富集在结合功能和催化活性功能,在细胞位置上主要富集在质膜和液泡。苎麻差异表达基因主要富集途径为谷胱甘肽代谢途径以及半胱氨酸、甲硫氨酸代谢途径,共有55个基因上调表达。更多还原

【Abstract】 With the development of mining, industry and agriculture, heavy metals pollution becomes more and more serious, and has threatened soil ecosystem, agricultural production and human health seriously. Phytoremediation is attracting interest and attention from governments and enterprises technique as a potentially engineering-economical, cost-effective, and green to clean up heavy metal from polluted soil.Ramie (Boehmeria nivea(L.) Gaud.), also named "China grass", is an important source of cellulose-rich natural fibres(bast fibres) which has anti-stress ability, rapid growth and higher biomass. Ramie’s primary purpose is making clothing, culturing ramie could minimizes the potential hazard of bringing toxic metals into food chains. It is significative to renovate heavy metals from contaminated soil with ramie due to its high accumulation and high economic value.In this study, heavy metal concentrations and bioaccumulation of ramie growing on 3 mining areas in Shimen, Lengshuijiang and Liuyang of Hunan Province were analyzed. Then, pot and field experiments were carried out to investigate the Cd tolerance of 9 ramie varieties, screen the tolereance index and analyse the capacity of ramie varieties to absorb Cd from contaminated soil. At the last, expression of Cd stress response genes were researched using solexa sequencing. The main results are as follows:1.Heavy metal concentrations and bioaccumulation of ramie growing on mining areasSoils from realgar mine area(Shimen) was heavily contaminated by As, while soils from antimony mine area(Lengshuijiang) was highly contaminated by Sb, and Qibao mountain area(Liuyang) was mainly contaminated by Cd. Ramie from these 3 sites grew well in different metal-contaminated habitats. In these 3 sites, Cd, As, Sb concentrations in ramie were 2-10,9.9-147.5,1.2-338 times higher than that in other plant species, respectively. And Sb, As concentration in ramie leaves were higher than that in roots and stems. Cd, As concentrations in ramie shoot were significantly correlated to Cd, As concentration in soil respectively. While Pb, Sb, Zn and Cu concentration in ramie shoot was not significantly correlated to Pb,Sb,Zn and Cu concentration in soil. Among these samples, the highest bioaccumulation factor(BF) and transfer factor(TF) of Cd were 2.07 and 3, those of As were 1.04 and 12.42, and those of Sb were 1.91 and 9.04,respectively. The total accumulations of Cd, Pb, As, Sb, Zn and Cu in these ramie samples were reached 0.11,1.17,0.72,7.97,6.71 and 1.69 kg/hm2 respectively. Ramie is a good candidate for phytoremediation of mining areas contaminated by multi-metals.2. Adaptability and tolerance evaluation of ramie varieties to CdHeight, diameter of stem, thickness of bark, biomass and chlorophyll of ramie were significantly reduced by Cd concentration in pot and field culture.Low Cd concentration enhanced the growth of some varieties of ramie, biomass of Shiqianzhugen,Yichun hongxin,and Chuanzhu No.1 which grown in Cd solution (46mg/L) were higher than that grown in control, biomass of Shiqianzhugen and Zhongzhu No.1 which grown on soil added with Cd(25mg/kg) were higher than that grown in control. The TI of Cd in ramie varies with varieties. The physiological and agronomic traits related to Cd tolerance of pot and field experiment were measured and analyzed, the results showed that screening ramie genotype by using height, biomass of shoot, biomass of root and chlorophyl of ramie grown in Cd solution as screening index would probably be an effective method to screen ramie genotype with high Cd tolerance. Nine ramie varieties were clusting 3 types by response to Cd, high-tolerance type, middle-tolerance and low-tolerance type.3. Uptake and accumulation of Cd in different ramie varietiesCd concentration in bark was significantly higher than that in other organs of ramie. When Cd concentrations were 23,46,91 and 182mg/L in solution respectively, Cd concentration in aboveground parts were 22.93,37.22,70.60 and 86.97mg/kg, which was significantly correlated to Cd concentration in solution. In field experiment, there were significant differences in Cd concentrations of aboveground part, under CK and Cd treatments(25,100mg/kg), Cd concentrations in shoot were 7.75,28.19 and 45.11mg/kg respectively. BF was decreased with the increase of Cd concentration. There were significance differences in Cd accumulation in shoot between Cd treatment and varieties. Two varieties were selected to renovate different pollution soil, Zhongzhu No.1 is fit for moderate contaminated soil by Cd(1-2mg/kg), Fushunqingma is fit for highly contaminated soil by Cd(25 and 100mg/kg). Nine varieties would class to four types by Cd concentration and tolerance:high-tolerance and low concentration, high-tolerance and high-concentration, low-tolerance and low-concentration, low-tolerance and high-concentration.4.Expression of Cd stress response genes in ramieTwo RNA libraries of Cd stress(100mg/kg) and control of ramie root were sequenced using solexa, and over 3.5 million short sequence reads were produced.3887 differently expressed genes of ramie root under Cd stress were selected referring to the significance of digital gene expression profiles. By blasting search to NCBI, we found most of differently expressed genes were unknown. The results of gene ontology functional enrichment analysis for differently expressed genes showed that main functions of these genes were binding and catalytic activity, and the main cell component which these genes enriched were plasma membrane and vacuole.The main pathways of these genes were glutathione metabolism and cysteine and methionine metabolism.更多还原