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题名: 叶际微生物传感器原位监视系统的研究
作者: 于清
学位类别: 博士
答辩日期: 2012
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 庄国强
关键词: 甲苯类有机物检测 ; 微生物全细胞传感器 ; the detection of BETX ; 叶际微生物生态位 ; 腺毛头 ; 生物膜
其他题名: BTEX monitoring system based on phyllospheric whole-cell biosensor
中文摘要:
       生物传感器,特别是微生物全细胞传感器在对环境中的污染物进行即时监控以及反映环境污染物的生物毒性方面的优势,近年来成为环境污染物环境监测方面的一个研究的热点。 微生物的种类十分丰富,能够代谢的底物种类更是无比的丰富。这一特性使微生物特别适合构建全细胞传感器。但是微生物个体微小,不利于在线监测分析。植物虽然缺乏像微生物那样的丰富的调控回路,但是植物叶子表面寄生着大量的微生物。我们能否利用植物叶际微生物构建全细胞传感器,并利用植物作为载体来构建用于监测环境污染物的原位监视系统? 本文就是基于这一设想,利用寄生于烟草植物叶面的微生物以能够检测甲苯类有机物的调控元件为模式构建微生物全细胞传感器。并利用植物为载体来研究这一系统应用于环境污染物监视以及食品安全方面可行性。 本文主要研究内容: 1.基于对恶臭假单胞菌对甲苯类有机物降解途径构的理解构建了能够检测该类物质的全细胞传感器。并针对构建传感器在信号强度,检测灵敏度以及检测时间上的缺陷,首次在翻译水平上对传感器报告基因的非翻译区基因元件进行了改造。改造大幅提高的细胞传感器的响应灵敏度,缩减了检测时间;利用mRNA定量分析的方法阐述了改造方法引起的报告基因mRNA的稳定性以及mRNA翻译效率的改变是传感器性能提高的原因。 2. 对烟草叶际可培养微生物进行了分离测序分析并对其进行了多样性分析。选择烟草叶际微生物-假单胞菌(Pseudomonas azotifigens)和草生欧文氏菌(Erwinia herbicola)进行了荧光蛋白标记并对他们的生态位进行了跟踪调查。经研究:首次证明了微生物能够在叶际环境中形成类似于水生生物膜的“三明治”结构。首次发现了腺毛头部可作为微生物生存的生态位。这些发现对于重新认识微生物在叶际环境中的生态位分化以及微生物微观生态将产生积极影响。另外我们发现微生物在叶子表面易生成生物膜结构且它们的分布与叶子的结构有着直接的关系。主要分布于植物的腺毛根部,叶脉的沟槽等叶面结构上。标记微生物的生存与相对湿度有着很大的关系,湿度降低会导致微生物大量死亡。 3. 构建了基于绿色荧光蛋白为报告基因的传感器质粒,并得到了很好的响应。并先后进行了以下尝试:利用基因组转座的方式将上述传感器元件转入微生物假单胞菌的基因组中。改造高信噪比的传感器元件与构建串联报告基因(5个),并进行将它们转座到叶际微生物假单胞菌中。
英文摘要:       Microbe-based whole cell biosensors were one of the focuses for the environmental study issue, because of their rapid in speed and cheap in price for on-line screening of general toxicity or stress. There are many kinds of metabolic pathways for microbes to metabolise the pollution in nature environment. So microbes can provide genetic motifs for the construction of whole cell biosensors. But microbes are very small in the scale of a single cell. So it is difficult to use microbe- based whole cell biosensors in visual study. In the environment, plants compose of a wide range of division. As other higher organisms, plants lack the metabolic pathways of microbe both in amounts and specificity. But on phyllosphere, there are a large number of microbes. Is there any possibility for us to construct phyllospheric microorganism-based whole cell biosensors and use the plant as the carrier for the environmental study? Based on this idea, in this dissertation, we constructed some whole cell biosensors responsing for BTEX and have tried to construct phyllospheric microorganism based whole cell biosensor to moniter the environmental stress and food security. The main contents of this dissertation include: First, we constructed a biosensor responsing for BTEX based the genetic motif from Pseudomonas putida (ATCC 33015). In order to make the biosensor fit for the visible dectection on phyllosphere, we modified the biosensors in the translation level at the first time. The modifications improved the sensitivity and reduced the detection time by a large margin. Using the method of QPCR, we analysed the relative amounts of mRNA from these whole cell biosensors and explained that the mRNA stability and translation effective are the reasons caused the change. Second, we analysed the diversity of culturable microbes from tobacco leaves. We marked the microbe Pseudomonas n5and Erwinia herbicola with fluorescent protein plasmid. Using these marked microbes, we studied the ecological niche of them. In this work, we first revealed that phyllospheric microorganisms can form sandwich-like biofilms on phyllosphere just as aquatic bacterial does and the head of the glandular trichome can act as habitats for microbes. These discoveries will give us a new sight for the study of the microecology. Third, we replaced the reporter gene of the biosensor plasmid by the gfp. The biosensor can act well in detection. We inserted the modified reporter modif into the genome of the Pseudomonas n5. The Pseudomonas n5- based biosensor did not show a good expression. So we replaced the single gfp gene with its five tandem duplications and the original reporter motif with a new one having a higher signal to noise ration (SNR). Finally, we inserted these motifs into the genome the genome of the Pseudomonas n5. But there are no positive results for this work.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35168
Appears in Collections:中科院环境生物技术重点实验室_学位论文

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Recommended Citation:
于清. 叶际微生物传感器原位监视系统的研究[D]. 北京. 中国科学院研究生院. 2012.
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