有机磷农药降解质粒在土壤中的转化规律研究

发布时间：2018-01-30 20:50

本文关键词： 土壤矿物胞外DNA分子吸附水平基因转移等温滴定微量热流式细胞术　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：土壤是地球上最大的生物多样性宝库,土壤微生物间的基因交流是提高生物适应环境和生存能力的重要途径,也是形成土壤生物群落多样性的遗传信息基础。随着遗传工程微生物向土壤环境中的释放呈日益增长的趋势,这些微生物及其携带的用以提高农作物产量或修复治理污染的遗传基因在环境中的安全及归宿引起了科学工作者及公众的广泛关注。本文以革兰氏阳性细菌枯草芽孢杆菌(Bacillus subtilis)、革兰氏阴性细菌恶臭假单胞菌(Pseudomonas putida)及多种典型土壤矿物为材料,综合应用等温吸附与解吸、等温滴定量热和衰减全反射红外光谱技术,测定了DNA与细胞-矿物复合物作用过程吸附量、亲和力、热力学参数及基团变化,以揭示其与土壤组分的相互作用机制。并以有机磷农药降解质粒为对象,结合荧光定量PCR和流式细胞术等方法,研究了游离态及固定态胞外DNA在环境中的存留时间,及向土著微生物进行水平转移的可能性。获得如下主要结果:(1)揭示了DNA在土壤细菌-矿物复合物上的吸附行为及作用机制。选取鲑鱼精DNA、两种常见的土壤细菌(枯草芽孢杆菌和恶臭假单胞菌),以及三种典型土壤矿物(高岭石、蒙脱石和针铁矿)为实验材料。表明所有细菌-矿物复合物对DNA的吸附可用Langmuir方程拟合。DNA在枯草芽孢杆菌-矿物复合物上的吸附量相比其在两种单一组分上的等比例吸附值之和有所提高,表明细菌与矿物的吸附能增加该体系对DNA的吸附量。DNA与枯草芽孢杆菌-矿物复合物结合时的焓变主要来自细菌与DNA分子,其主要作用力为范德华力及氢键。DNA与恶臭假单胞菌-矿物复合物相互作用的情况则受到矿物类型的影响,其与恶臭假单胞菌-高岭石复合物进行吸附时主要作用力为范德华力及氢键;而与恶臭假单胞菌-蒙脱石/-针铁矿的作用力则应归结于脱水作用及水分子重排引起的熵增。(2)成功构建携带双重标记的有机磷农药降解质粒,发现游离及固定态质粒的转化效率受质粒类型、矿物种类及宿主类别等多因素影响。以有机磷高效降解质粒pB17、广宿主载体pBHR1及抗重金属细菌恶臭假单胞菌X4的小质粒为材料,对其进行增强型绿色荧光蛋白基因egfp及卡那霉素抗性基因kmr双重标记,得到重组质粒pRB17、pXr及pBr。并对三种游离态质粒及与蒙脱石、高岭石、针铁矿吸附后的固定态质粒进行了实验室水平的转化实验。结果表明,以广宿主载体为基础所构建的降解质粒具有向更多种类细菌转移的能力。游离态的DNA较固定态DNA有更高的降解速率。DNA与矿物吸附后进行转化,所获得转化子的数量有不同程度的降低。游离态pBr转化大肠杆菌DH5α其转化效率可达2.98×105个转化子/μg DNA,而与蒙脱石、高岭石及针铁矿吸附后,转化效率分别下降了2~4个数量级。类似的结果也出现在pXr转化DH5α的过程中,与三种矿物结合后的转化效率下降了1~3个数量级。表明与DNA亲和力强的矿物降低DNA转化效率的作用更明显。(3)阐明了游离及固定态质粒在土壤环境中的稳定性特征及生物可利用性。结果表明DNA在进入土壤的最初阶段降解迅速,3~7天后降解曲线逐渐平缓。游离态DNA较固定态DNA在土壤中的降解速度更快,经DNA回收转化大肠杆菌后,其转化效率的下降也更为显著。DNA被不同类型矿物吸附后其降解效率也存在差异。与高岭石和针铁矿相比,吸附于蒙脱石表面的DNA分子降解速率更慢,在第3天时残留量仍保持在50%以上,而且第15天仍能成功转化至大肠杆菌中。表明蒙脱石对DNA具有更好的保护作用,使其能免受核酸酶的降解,具有更长时间的生物可利用活性。(4)比较了多种检测环境中小概率水平基因转移事件的实验手段,提出抗生素富集联用流式细胞分选术能有效分离土壤中的罕见目标细胞。证实了胞外DNA具有向土著微生物进行转移的能力。选择游离态及蒙脱石固定态质粒pBr及pXr作为土壤转化实验DNA供体,武汉市两处地点的棕红壤作为受体细菌来源,土样样品设置堆肥或有机磷农药处理组。利用荧光显微镜、标记基因PCR扩增及流式细胞术直接对土壤中的转化子进行检测,检测限均低于2×10-6,未能检测到阳性转化子的存在。通过抗生素富集及流式细胞多轮分选技术对样品进行筛选可有效将检测限提高至10-11量级。所分得菌株均为来源于菜园土和添加农药处理实验组的pBr质粒转化子,属肠杆菌目肠杆菌科。广宿主载体的使用、土壤微生物群落组成与结构及农药施用对土壤菌群的选择性压力或为转化效率增加的重要原因。
[Abstract]:The soil is the world's largest repositories of biodiversity, genetic exchange between soil microorganism is an important way to improve biological adapt to the environment and the ability to survive, is the formation of genetic information based soil biological diversity. As to the genetic engineering of microorganisms in soil environment showed a growing trend, and used to carry these microorganisms to improve the safety and fate of genetic crop or repair pollution in the environment and attracted extensive attention from scientists and the public. The gram positive bacterium Bacillus subtilis (Bacillus subtilis), gram negative bacteria (Pseudomonas putida) and a variety of typical soil mineral materials, comprehensive application of adsorption and desorption the isothermal and isothermal titration calorimetry and attenuated total reflection infrared spectroscopy, DNA and cell - mineral complex function determination The process of adsorption, affinity, thermodynamic parameters and structure change, and to reveal the interaction mechanism of soil components. And the organophosphorus pesticide degrading plasmid as the object, combined with fluorescence quantitative PCR and flow cytometry methods on the retention time of free and immobilized DNA in the extracellular environment. And to the possibility of horizontal transfer of indigenous microorganisms. The main results are as follows: (1) reveals the adsorption behavior and mechanism of DNA in soil bacteria mineral complexes. Selection of salmon sperm DNA, two kinds of common soil bacterium (Bacillus subtilis and Pseudomonas putida), and three kinds of typical soil minerals (kaolinite, montmorillonite and goethite) as experimental materials. The adsorption amount showed that all bacteria mineral complexes on the adsorption of DNA can be simulated with Langmuir equation.DNA in Bacillus subtilis - mineral complexes compared to their two single in group On the proportion of adsorption and increased adsorption of bacteria and minerals can increase the system for the adsorption of DNA on.DNA and Bacillus subtilis - mineral complexes with the enthalpy change mainly from bacteria and DNA molecules, the main force is the interaction force and hydrogen bond, van Edward and stench Pseudomonas.DNA gingivalis - mineral complexes is influenced by the type of mineral, the Pseudomonas putida kaolinite composites were mainly adsorption force is the force and hydrogen bond and van Edward; force and Pseudomonas putida - montmorillonite / goethite should be attributed to the dehydration effect and water molecular rearrangement caused by entropy. (2) the successful construction of organophosphorus pesticide degrading plasmid carrying double labeling, found that the conversion efficiency of free and immobilized plasmid by plasmid type, influencing factors of mineral species and host types. With efficient degradation of organic phosphorus Plasmid pB17, plasmid small broad host vector pBHR1 and heavy metal resistant bacteria Pseudomonas putida X4 as materials, the enhanced green fluorescent protein gene EGFP and kanamycin resistance gene KMR double labeling, recombinant plasmid pRB17, pXr and pBr. and three kinds of free plasmid and montmorillonite, kaolinite. Immobilized plasmid goethite adsorbed by transformation experiment laboratory level. The results showed that the broad host vector for degradation plasmid built has the ability to transfer more species of bacteria. The free state is DNA fixed DNA has a higher degradation rate of.DNA and after adsorption transformation quantity obtained transformants were decreased. Free pBr is transformed into Escherichia coli DH5 alpha and its conversion efficiency is up to 2.98 x 105 transformants / g DNA, and montmorillonite, kaolinite and goethite after adsorption, the conversion efficiency respectively. Decreased by 2~4 orders of magnitude. Similar results also appeared in the process of transforming DH5 alpha pXr, and three kinds of minerals with conversion efficiency decreased after 1~3 orders and DNA. Show strong affinity of mineral reduced DNA conversion efficiency effect is more obvious. (3) to clarify the biological characteristics and stability of free and immobilized plasmid in soil availability. The results showed that DNA was degraded rapidly in the initial stage into the soil, 3~7 days after the degradation curve becomes slower. The degradation rate of free DNA was immobilized in the soil DNA faster, DNA recovery was transformed into Escherichia coli, the conversion efficiency decreased more significantly.DNA is different after adsorption and the degradation efficiency are also different. Compared with kaolinite and goethite, DNA molecules adsorbed on montmorillonite surface degradation rate more slowly, the residues in third days remained at more than 50%, and fifteenth days Can be successfully transformed into Escherichia coli. That has better protective effect of montmorillonite on DNA, which can degrade from nuclease, with longer bioavailable activity. (4) compared a variety of testing environment for small and medium sized probability of horizontal gene transfer experiments of events, a combination of antibiotics concentration flow cytometry the operation can effectively separate rare target cells in soil. Confirmed that extracellular DNA has the ability to transfer to the indigenous microorganisms. Selection of free and immobilized montmorillonite plasmid pBr and pXr as the experimental soil DNA donor, Wuhan city locations in the brown red soil as the receptor sources of bacteria, soil sample set compost or organophosphorus pesticide treatment group. Using fluorescence microscopy, the marker gene was amplified by PCR and flow cytometry detected directly in transformants in the soil, the detection limit is lower than 2 * 10-6, failed to detect a positive turn The son's existence. Based on the samples of antibiotic enrichment and flow cytometry sorting technology can be effective in screening rounds will be increased to 10-11 magnitude. The detection limit of isolated strains were derived from vegetable soil and add pesticide pBr plasmid treatment group transformants, genus enterobacteriales Enterobacteriaceae. The use of broad host vector, composition and structure of soil microbial community and pesticides on soil microflora or selective pressure as an important reason for the increase in conversion efficiency.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X592

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