【作者】 王湘；

【导师】 朱咏华；

【作者基本信息】 湖南大学 ， 生物学， 2019， 硕士

【摘要】 重金属污染已经成为全世界关注的环境问题,其中镉污染严重危害了农作物的生长和人体的健康。对污染生境的修复,微生物的作用与传统的物理化学方法相比,更加安全和高效,具有很大的开发潜力。实验室前期从铅锌尾矿区的杨树根部分离得到了一株内生细菌Burkholderia cenocepacia YG-3,该菌对铅、锌、镉的耐受能力都很强。为了探究B.cenocepacia YG-3的耐镉机制,本论文主要从以下方面开展了研究:(1)YG-3在镉胁迫下的表征:在含100 mg/L和500 mg/L Cd的LB培养基中分别培养60 h和7 d,检测YG-3对Cd的吸收和吸附情况,结果显示100 mg/L Cd对YG-3生长影响较小,菌体采取两种策略富集Cd,即吸收了17.9%的Cd,表面吸附了29.2%的Cd;500 m/L Cd条件下,主要采取表面吸附策略富集Cd,占Cd总量的31.8%。通过环境扫描电镜(SEM),X射线能谱分析(EDX),透射电镜(TEM),红外光谱(IR)手段分别对菌体表面形态、细胞元素、菌体内部形态及细胞组分的变化进行表征。发现菌体在100 mg/L Cd胁迫下形态无明显变化,在500 mg/L Cd胁迫下菌体表面有大量Cd沉积物,且与Cd结合的细胞组分主要为多糖和蛋白质。(2)YG-3在镉胁迫下的生理生化特性:检测YG-3在0、100和500 mg/L Cd浓度下分泌的铁载体与胞外多糖产量,结果表明Cd能够诱导YG-3显著提高胞外分泌物的产量。在100 mg/L Cd条件下,铁载体与胞外多糖产量比对照组都提高了1.4倍,在500 mg/L Cd条件下,比对照组分别提高了1.8和2.1倍。(3)YG-3在镉胁迫下的定量蛋白质组学特点:运用label-free定量蛋白质组学方法,分析YG-3对照组与500 mg/L Cd处理组的差异表达蛋白。分析表明,YG-3存在复杂多样的耐镉机制,包括增加胞外分泌的铁载体与多糖的产量在胞外与Cd结合;提高细胞壁组分的合成,保证Cd被吸附在细胞壁而被阻挡在胞外,减少Cd离子的毒害;提高金属蛋白等与Cd亲和力高的组分的产量,在胞内与Cd结合,减少Cd与功能性蛋白的结合;增强离子外排泵的作用将进入胞内的Cd主动运输至胞外,来减少胞内Cd的含量;通过提高抗氧化物的合成,缓解Cd造成的过氧化胁迫等。本论文探究全面阐明了细菌YG-3对Cd耐受的机制,完善了细菌抗重金属污染的机理,为将来应用该菌或与杨树联合作用修复镉污染打下基础。更多还原

【Abstract】 At present,heavy metal pollution has become an important environmental problem all over the world.Excessive cadmium seriously endangers the growth of crops and human health.Compared with the traditional physical and chemical methods,using the bacteria from contaminated habitats is safer and more efficient.In our previous study,an endophytic bacteria YG-3 was isolated from the root of poplar taken from the leadzinc tailing area,and showed strong tolerance to lead,zinc and cadmium.It was identified as Burkholderia cenocepacia.In order to get integrated sight into the cadmium resistance mechanism of B.cenocepacia YG-3,this study was carried out in the following aspects:(1)Characteristic tests of YG-3 under Cd stress: the adsorption of YG-3 at 100 mg/L and 500 mg/L Cd was detected,and characterized by environmental scanning electron microscopy(SEM),X-ray energy spectrum analysis(EDX),transmission electron microscopy(TEM)and infrared spectroscopy(IR).The results showed that 100 mg/L Cd had little effect on the growth of YG-3.The bacteria adopted two strategies to enrich Cd,in which 17.9% was absorbed and 29.2% was adsorbed on the surface.Under the condition of 500 m/L Cd,YG-3 mainly adopted the surface adsorption strategy to accumulate Cd,accounting for 31.8%.In addition,the morphology of the bacteria did not change significantly under 100 mg/L Cd exposure,while a large number of deposits containing Cd were detected on the surface of the bacteria in 500 mg/L condition,and the cell components bound to Cd were mainly polysaccharides and proteins.(2)Physiological and biochemical characteristics of YG-3 under Cd stress: the yield of siderophores and exopolysaccharide secreted by YG-3 at different Cd concentrations were detected.The results showed that Cd could induce YG-3 to significantly increase the yield of these complex.Under the condition of 100 mg/L Cd,the yields of siderophores and exopolysaccharide were 1.4 times higher than those of the control group,and 1.8 and 2.1 times higher than those of the control group at 500 mg/L Cd,respectively.(3)Quantitative proteomics study of YG-3 under Cd stress: the label-free quantitative proteomics method was performed to analyze the differentially expressed protein between the control group and the 500 mg/L treatment group,and the integrated cadmium tolerance mechanism of YG-3 was obtained,including the secretion of siderophores and exopolysaccharides bound to Cd;improving the synthesis of cell wall components,ensuring that Cd is adsorbed on the cell wall and blocked outside the cell wall;increasing the production of components with high affinity to Cd,such as metalloproteins,to bind with Cd in the cell,thus reducing the binding of Cd to functional proteins;intracellular Cd is actively transported to the outside of the cell by efflux pumps to reduce the content of intracellular Cd;alleviate the peroxidation stress caused by Cd by increasing the synthesis of antioxidants.In this paper,the specific and comprehensive tolerance mechanism of YG-3 under Cd stress was explored.These results provide clues for the future study on the combined effect of poplar to remediate cadmium pollution,and laying a foundation for the practical application of this strain.更多还原