【作者】 高欢欢；

【导师】 赵惠燕；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2013， 博士

【摘要】 在农药和化肥过量使用、城市污水等大量排放以及金属矿业飞速发展等人类活动的强烈影响下,重金属污染已经成为最严重的全球环境问题之一,威胁着生态系统的平衡与发展。前人已经进行了大量有关短期重金属胁迫的研究,证明重金属对生物生长发育繁殖有显著影响,但重金属长期胁迫影响和机理如何,尚不清楚。麦长管蚜(Sitobion avenae)是重要的农业害虫,其典型的R对策特征,使其能快速适应环境而进化,成为应对环境变化的很好的实验材料,因此,研究重金属长期胁迫下蚜虫防御机制以及适应机理不仅具有重要的理论意义而且在防治农业害虫方面具有重要的应用价值。本研究在实验室条件下,模拟麦长管蚜种群在重金属胁迫条件下,研究短期Cd和Zn处理后麦长管蚜生长发育和种群的变化、长期胁迫下麦长管蚜体内抗氧化酶体系、羧酸酯酶(CarE)和乙酰胆碱酯酶(AchE)的活性和基因表达规律,从而探索麦长管蚜在长期Cd和Zn胁迫下的防御机制。主要研究结论如下：1、小麦对土壤中重金属的吸收呈Holling Ⅱ型规律,即吸收量随土壤中Cd和Zn含量的增加而增加,当达到一定剂量时,植物的吸收量趋于稳定。根据Holling Ⅱ得出,其最大吸收量分别为97.087mg/kg和1000mg/kg。由此可知,在一定的含量范围之内,寄主植物会积累土壤中的重金属。2、Cd短期胁迫对麦长管蚜种群参数有显著影响：净增殖率(R0)、内禀增长率(r)和周限增长率(λ)有所下降,并且在20mg/kg的处理下最低。表明Cd短期胁迫抑制麦长管蚜的生存和繁殖；20mg/kg的浓度处理为敏感阂值。3、Cd长期胁迫对麦长管蚜体内超氧化物歧化酶(SOD)活性、基因相对转录水平有显著影响,随着世代的增加呈现先上升后下降的趋势；连续处理5代时,酶活性和基因表达水平受到抑制,Cd浓度高于40mg/kg时,抑制作用更明显；第10代的时候均高于对照,但是在10代之后,不仅酶活性受到抑制,基因的转录也从上调模式转变为下调模式。表明：40mg/kg的Cd是影响酶活性和基因表达的阈值剂量；重金属胁迫影响的关键世代是第10代。4、Cd长期胁迫对麦长管蚜体内解毒酶——羧酸酯酶CarE活性和基因的相对转录水平有显著的影响,随世代增加表现出先升高后降低的趋势。CarE基因表达在10代时出现上调模式；处理至20代的时候,高浓度Cd胁迫下,CarE活性与对照已无显著差异。表明：重金属Cd提高了CarE活性和基因表达水平；低剂量Cd胁迫下,CarE发挥了很重要的解毒作用。5、Zn长期胁迫对麦长管蚜体内SOD活性和基因相对转录水平有显著影响：随着世代增加,Zn对酶活性的影响逐渐下降,到30代时已经与对照无显著差异；而SOD基因相对转录水平在较低世代时呈现上调的表达模式,其中第5代,高浓度的Zn增加基因表达水平会更显著。但是15代之后,麦长管蚜对1600mg/kg的Zn的敏感性下降,在25和30代时已经与对照无显著差异。6、Zn长期胁迫条件下,麦长管蚜体内CarE活性受Zn浓度和处理世代显著影响：低世代酶活性有所升高,但随着世代增加,活性逐渐下降；高于15代之后,高浓度的Zn则抑制CarE的活性。CarE基因表达模式与对照相比呈现出上调模式,但到高世代,上调现象并不显著。表明,CarE可以作为麦长管蚜对重金属产生调控及适应的生物指标,在选择压力长期存在的情况下,会有自身特定的基因表达模式。7、重金属Cd长期胁迫下,麦长管蚜体内POD和CAT活性受到了显著地影响：Cd在所有世代中均抑制了POD活性；连续处理低世代抑制了CAT酶的活性,随着世代的增加,表现出先升高后降低的趋势,虽然在15代时其活性有所升高,但到20代时仍然受到抑制。重金属Zn长期胁迫下,15代之前的麦长管蚜体内POD和CAT活性均显著增加,高浓度的Zn诱导作用更明显,但随着世代增加均有递减的趋势,在30代时,Zn对POD的活性已无显著影响,CAT的活性甚至低于对照。因此,短期Cd和Zn处理之后,麦长管蚜会通过提高抗氧化酶的活性来防御重金属的危害,但长期高剂量胁迫,其解毒作用会受到限制。8、Cd和Zn长期胁迫下,麦长管蚜体内AchE基因表达水平随Cd和Zn处理时间和浓度的增加有先上升后下降的趋势。Cd和Zn连续处理低世代均抑制了AchE表达;而Cd处理10代之后,基因表达出现上调模式,但是达到20代连续处理时,表现为下调模式；40mg/kg是Cd胁迫下转变表达模式的关键浓度,与SOD和CarE的关键浓度一致；而Zn胁迫15代是关键世代,高于15代后,基因表达出现显著的下调模式。表明：乙酰胆碱酯酶的防御作用在10-15代时发挥了最有效的作用,但是随着世代的增加,其水解作用受到了限制。综上所述：土壤重金属Cd和Zn通过小麦显著影响麦长管蚜的生长发育和种群数量；诱导麦长管蚜体内抗氧化酶、羧酸酯酶、乙酰胆碱酯酶的表达活性,补偿重金属造成的损害。长期重金属胁迫导致蚜虫体内毒性剂量不断的增加,限制了体内抗氧化酶、羧酸酯酶、乙酰胆碱酯酶的防御作用,表明,长期胁迫使蚜虫自身产生了新的适应机制以应对自然选择和维持正常的代谢。更多还原

【Abstract】 The concentrations of heavy metals in the topsoil can increase with the addition of chemical fertilizers, pesticides, and sewage from urban and metal mill. Heavy metal contamination from these sources has become an important global environmental problem, which threatens the ecological equilibrium. Effect of heavy metal stress on insects under the short-time exposure is involved in the previous researches. However, under the stress of heavy metal for long time, the research on defense mechanism of insects is not enough at present. As the destructive pest throughout the world, Sitobion avenae can be the good material of researching adaptation and evolution of insects as their biological characteristics of parthenogernesis and high fecundity. Therefore, the exploration about defense mechanism of S. avenae exposed to heavy metal for long time has the theoretical and practical significance for the integrated pest management.In the simulative natural environment, this study evaluated the population dynamic of5. avenae. The activity and gene expression patterns of antioxidant enzymes, carboxylesterase (CarE) and acetylcholin esterase (AchE) were carried out under the long-term exposure of Cd and Zn. We aimed to explore the defense mechanism of S. avenae under the stress of heavy metal. The following are the conclusions:1) The result of estimating concentration in wheat showed that the concentration of Cd and Zn increased with the increasing of that in soil. When it reached a certain concentration, the absorbed dose in wheat would be saturated. According to Holling Ⅱ functional response, the largest absorbed doses of Cd and Zn in wheat were97.087mg/kg and1000mg/kg respectively. It was concluded that host plant would accumulate heavy metal from contaminated soil within a certain range.2) The population dynamic of S. avenae was evaluated under the short-term exposure of Cd. The result showed that the survival rate and fecundity were reduced in the later stage of aphids. The life table parameters, such as the net reproductive rate (Ro), innate capacity of increase (r), and finite rate of increase (λ), were also inhibited by Cd. The most significant influence was occurred at the concentration of20mg/kg. This concentration was more sensitive for S. avenae. 3) The effect on activity and the transcript level of superoxide dismutase (SOD) gene of S. avenae were estimated under the exposure of Cd for20generation consequently. It was resulted that, they were inhibited at5th generation, of which this inhibition was more obvious at high concentration of Cd. Although the SOD activity and gene expression of S. avenae were increased at10th generation, they were still inhibited after treated for10generation. SOD gene of S. avenae was more sensitive at the concentration higher than40mg/kg. Therefore,40mg/kg and10th generation were the critical dose and stage for S. avenae to transform gene expressive pattern from up-regulation to down-regulation4) The effect on CarE activity and gene expression of S. avenae were estimated under the exposure of Cd for20generations consequently. The CarE activity and transcript level were induced by Cd in10th generation at all the concentration. However, the higher concentration than40mg/kg of Cd had no significant effect on the CarE activity and gene expression in20generation.40mg/kg and10th generation were the critical dose and stage. It was concluded that, under the stress of low-level of Cd, CarE played an important role of resisting damage, and this role of detoxification was limited under the high-level Cd.5) The effect on the activity and transcript level of SOD gene of S. avenae were estimated under the exposure of Zn for30generations consequently. SOD activities of all the treatments within the generation were higher than the control, but decreased with increasing of generation treated significantly. The expression of SOD gene was up-regulated, of which5th generation was more significant in1600mg/kg. However, the sensibility was reduced after15generations for1600mg/kg of Zn. No difference was observed for SOD activity and gene expression in the30th generation of aphids. Therefore, the antioxidation of SOD was lost under the long-term exposure of Zn with high concentration.6) The effect on CarE activity and gene expression of&avenae were estimated under the exposure of Zn for30generations consequently. The CarE activity and transcript level were induced by Cd in the lower generations, but decreased with increasing of generation treated significantly.1600mg/kg of Zn inhibited CarE activity after20generations and no difference was found for transcript level. It was conclude that CarE could be the sensitive biomarker for S. avenae response to the presence of Zn. In order to adapt to the stress of heavy metal Zn, the S. avenae had the particular patterns of gene expression under the long-time stress.7) The effect on activity of peroxidase (POD) and catalase (CAT) of S. avenae was estimated under the exposure of Cd and Zn for long term. It was found that the activities of POD were reduced by Cd and no significant difference of POD activity was found in all generations. Under the stress of Cd, the peak value of CAT activity was occurred in15th generation, after that, it was decreased and lower than the control. The activity of POD and CAT of S. avenae was still affected by Zn significantly. They have the tendency of decrease with the increasing of generations. When aphids were treated for30generations, no obvious influence had been found on POD activity, CAT activity was inhibited by Zn evenly. The results demonstrated that S. avenae would maintain the metabolic balance through inducing antioxidant enzyme, but could not at the high-level toxicity.8) The effect on AchE gene expression of S. avenae under the exposure of Cd and Zn for long term:gene expression of AchE was induced and then reduced with the increasing of generation and concentration of Cd and Zn. At5th generation, gene expression of AchE was down-regulated by Cd and Zn. Under the stress of Cd for10generations, the expressive pattern was transformed to up-regulated, but still inhibited by long-term exposure of Cd (20generations).40mg/kg and10th generation were the critical dose and stage, which was inconsistent with SOD and CarE. However, the critical stage was15th generation for S. avenae under the stress of Zn. The expressive pattern was down-regulated after15th generation. It was concluded that, the resisting role of AchE to heavy metals was limited under the high-level toxicity.In conclusion, the development and population parameters of S. avenae were inhibited under the exposure of Cd and Zn though the accumulation in wheat. But S. avenae could adapt and compensate the toxicity of low-level heavy metals through inducing the expression of antioxidant enzymes, CarE and AchE. However, the physiological confusion would occur under the high-level stress. Under the long-term exposure of heavy metals, the defense of antioxidant enzymes, CarE and AchE would be restricted due to the high-level toxicity. Maybe the new advanced pattern of adaptation emerged更多还原