【作者】 刘伟；

【导师】 朱鲁生；

【作者基本信息】 山东农业大学 ， 农药学， 2009， 博士

【摘要】 持久性有机污染物(简称POPs),被认为是21世纪影响人类生存与健康的重要环境问题。由于持久性有机污染物在全球普遍存在,具有生物累积性、难以降解、可远距离传输、致癌致突变性和内分泌干扰等特性,它们所引起的环境与健康问题已经引起国际环境保护组织、各国政府和民众的高度关注。硫丹为有机氯杀虫剂,具有触杀和胃毒作用,杀虫谱广,广泛用于棉花、果树、蔬菜、茶叶等作物上,我国已大面积使用。20世纪90年代以后,越来越多的国家认识到了硫丹广泛使用的危害性,许多国家开始禁用或严格限制使用。作为一种即将被列入POPs名单的污染物,硫丹对人体健康和生态环境的潜在影响问题在国内外备受关注,研究其生态毒性非常重要。鉴于土壤微生物、动物、植物在生态系统中的重要性,本研究利用分子生物学技术,采用微核实验、彗星试验和变性梯度凝胶电泳实验,研究了硫丹对蚕豆、白三叶草、蚯蚓和土壤微生物的毒性效应,评价了硫丹对生物DNA的损伤作用和土壤微生物群落多样性的影响,对于了解和认识硫丹对生态系统的影响以及为建立快速、有效、实用的生物监控和测试技术具有重要意义。主要研究结果如下:1.为了探明硫丹对蚕豆的遗传损伤作用,采用毒理学实验方法,研究了0.1、1.0、10.0 mg/L浓度的硫丹对蚕豆根尖细胞有丝分裂、染色体畸变和微核的影响。研究结果表明,硫丹抑制蚕豆根尖细胞的有丝分裂。经硫丹处理后,蚕豆根尖细胞有丝分裂指数则逐渐降低,降低的程度随硫丹的浓度和染毒时间的增加而增强。随着硫丹的浓度和染毒时间的增加,蚕豆根尖细胞的染色体畸变率和微核率不断提高。有丝分裂指数与染色体畸变和微核率都呈负相关,染色体畸变率与微核率呈正相关,并具有明显的剂量-效应关系。在本试验条件下硫丹达到一定剂量浓度会对蚕豆根尖细胞造成DNA损伤,具有明显的遗传毒性。2.单细胞凝胶电泳实验(彗星试验)是一种快速、简单、直接的DNA损伤研究手段,能广泛应用于多种组织来快速检测环境基因毒性物质。本文通过彗星试验测定了硫丹对蚯蚓体腔细胞和白三叶草叶片细胞DNA的损伤作用。试验结果显示,在设定的四个检测时间(7d、14d、21d、28d),经不同浓度硫丹处理,蚯蚓体腔细胞和白三叶草叶片细胞DNA均有损伤,而且随着浓度的增加和时间的延长,彗星拖尾长度、Olive尾矩的值均有上升的趋势,这说明DNA损伤程度与硫丹的浓度和处理时间均有效应关系。表明硫丹对蚯蚓和白三叶草造成了基因损伤。本研究首次利用广泛分布的白三叶草作为受试生物材料,进行污染物的彗星试验检测试验。通过实验发现,和对照处理相比,经过硫丹处理后白三叶草叶片细胞能够通过彗星试验检测DNA损伤,而且随硫丹处理浓度的增加和暴露染毒时间的延长,细胞核产生的伤害程度也随之增加,具有较高的灵敏性。表明白三叶草叶片能应用于植物彗星实验,研究环境污染对植物DNA的损伤。3.采用三种不同方法,分别对土壤微生物总DNA进行提取效果的比较,结果发现运用化学试剂和酶结合的策略能够获得高质量、高产量的基因组DNA,较其他方法具有明显的优势和效果,并且获得的产物经纯化后更适用于随后的分子生物学实验。4.采用扩增细菌16Sr DNA V3区的通用引物,通过改变扩增反应体系中的Mg2+、dNTP、引物的条件,并采用Nest-PCR方法,优化了含有“GC”结构引物的PCR扩增方法,获得了较好的扩增产物,使得DGGE电泳图谱条带更清晰,可分辨的条带数量也更多。5.利用DGGE指纹分析技术研究了硫丹对土壤微生物群落多样性的影响。通过对不同培养时间(7d、14d、21d、28d)、不同浓度(0.1、1.0、10.0 mg/kg)硫丹污染下土壤微生物DNA进行DGGE多样性的分子指纹图谱分析,发现随着培养时间不同,各处理之间的土壤微生物群落多样性出现了一定的差异。在硫丹作用下,土样中一些细菌种群的生长受到抑制,随着硫丹或作用时间的提高或延长,其相关条带由深变浅,甚至消失。而另一些细菌种群的得到富集,其条带由无到有。在整个试验过程中,随着硫丹浓度的升高,与对照相比各泳道条带间的Jaccard指数相似性相应降低,在一定浓度和作用时间范围内,硫丹对土壤细菌种群的改变的影响存在剂量-效应关系。表明硫丹对土壤微生物群落结构有着明显的影响。更多还原

【Abstract】 Persistent organic pollutants (POPs) are a family of organic pollutants that are persistent, bioaccumulative aneasily cause adverse effects on human and organisms. POPs have been the concern of all over the word. They present immunotoxicity, endocrinetoxicity, reproductive and developmental toxicity, carcinogenesis and so on. The ecotoxicity studieon POPs should be strengthened.Endosulfan is one of the most commonly used organochlorine insecticides. It is being extensively used in crops field due to its broad spectrum of activity and relatively low cost. As a result of its widespread use, endosulfan has been ubiquitously detected in atmosphere, soils, sediments, surface water, rainwaters and foodstuffs.Taking into account the magnitude of use, environmental levels and human and ecological effects of this compound, many countries have recognized the hazards of wide application of this pesticide. Today, Endosulfan has been banned or severely restricted in over 30 countries, but it continues to be widely used in some developing countries like China and Thailand .Studies concerning effects of endosulfan on soil organisms are needed in order to evaluate its toxicity. The aim of the present study was to assess the genotoxic effects of endosulfan in Vicia faba, earthworm (Eisenia foetida), white clover (Trifolium repens L.) and soil microorganism. From molecule level, DNA damage and effects on soil microbial diversity induced by endosulfan were evaluated. Our objective was to get a more comprehensive understanding on the effects of endosulfan and provide more information about the potential ecological risk of this pesticide on the soil ecosystem.The main results are showed as follows:1. Vicia-icronucleus test is applied in most fields, as a detecting means. In order to prove up the influence of endosulfan towards Vicia faba, cell mitosis,chromosomal aberrations and micronucleus were researched. The results showed that when seeds were treated with different endosulfan concentrations, 0.1, 1.0 and 10.0 mg/L, the cell mitosis of seeds were inhibited.With the increasing of endosulfan concentration and poisoned time,the inhibited effect were both enhanced. With the increasing of endosulfan concentration and poisoned time, the frequency of the chromosomal aberration and micronucleus rate increased. There were significant influences on the chromosomal aberrations and micronucleus of seeds treated with endosulfan, which effects were relative to endosulfan concentration and poisoned time.It was very clear that this pesticide with a certain concentration had effects on cytogenetical toxicity of Vicia faba root tip cells.2. The comet assay has been widely used in the fields of genetic toxicology and environmental biomonitoring, which was a new tool for genotoxicity study. In the present study, earthworms were exposed with endosulfan concentrations of 0.1, 1.0 and 10.0 mg/kg in the soil. The white clover roots were immersed in hydroponic pots containing nutrient solutions of different endosulfan concentrations, 0.1, 1.0 and 10.0 mg/L. Tissues of each treatment were collected on the 7th, 14th, 21st and 28th days of treatment process. Significant effects from both concentrations and time of exposure were observed. Under our experimental conditions, the induction of DNA damage in Eisenia foetida and T.repens specimens appeared to be dose-dependent throughout the exposure period. And endosulfan induced DNA damage on earthworm and white clover nuclei.In this research, the potential for DNA damage, as measured by the Comet assay, to act as a biomarker of genetic toxicity in white clover plants was considered. A significant increase of DNA migration was observed on the nuclei obtained from T.repens leaves. During the exposure period, DNA damage increased as the exposure concentrations of the pesticide increased and DNA migration showed a dose-response relationship. The T.repens comet assay is as sensitive as the comet assay using earthworm cells which could be used in genotoxicity study of environmental pollutants. To our knowledge, this is the first report describing the use of T.repens for genotoxicity assessment of environmental pollutants using the comet assay. Based on experimental evidence obtained, we can suggest that DNA damage of leaf cells of T.repens may potentially be used as a bioindicator for detecting genotoxicants in ecosystem.3. In our study, three soil DNA extraction methods which based on different lytic principles for isolation of the total farmland microbial DNA were compared. The results showed that the chemiea-enzymatic method was better than other methods. The chemiea-enzymatic method could obtain the best DNA with the highest molecular weight and purity,which was more propitious to molecular ecologystudy. 4. The PCR method ,as there are“GC clamp”with the primer,was optimeized through the change of Mg2+、dNTP、primers. Primer,The traditional PCR and Nest-PCR strategy were compared. The result showed that with the Nest-PCR strategy,the aim gene fragment was best amplified. It was more suitable for DGGE study.5. DGGE method was applied to analyse the relative genetic complexity of microbial communities in soil treated with different endosulfan concentrations, 0.1, 1.0 and 10.0 mg/kg. The target gene was separated by DGGE,and the cluster analysis wascarried. From the result we can see that there were abundant bacterial communities at the different treated soil. The result showed that there were a certain differences in baeterial communities diversity in soil treated with different concentrations of endosulfan. The results showed that many bacteria were inhibited in soil treated with endosulfan. The Jacarrd indexes among the different samples and control were reduced throughout the exposure period. With the increasing of endosulfan concentration and poisoned time, some bands had been changed. It means that the mutual benefit communities were developed and others were restrained or disappeared. From the result we also can got the conelusion that DNA sequence of soil microorganism was affected by endosulfan.更多还原