本文關(guān)鍵詞： 土壤礦物 胞外DNA分子 吸附 水平基因轉(zhuǎn)移 等溫滴定微量熱 流式細(xì)胞術(shù)　出處：《華中農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：土壤是地球上最大的生物多樣性寶庫,土壤微生物間的基因交流是提高生物適應(yīng)環(huán)境和生存能力的重要途徑,也是形成土壤生物群落多樣性的遺傳信息基礎(chǔ)。隨著遺傳工程微生物向土壤環(huán)境中的釋放呈日益增長的趨勢,這些微生物及其攜帶的用以提高農(nóng)作物產(chǎn)量或修復(fù)治理污染的遺傳基因在環(huán)境中的安全及歸宿引起了科學(xué)工作者及公眾的廣泛關(guān)注。本文以革蘭氏陽性細(xì)菌枯草芽孢桿菌(Bacillus subtilis)、革蘭氏陰性細(xì)菌惡臭假單胞菌(Pseudomonas putida)及多種典型土壤礦物為材料,綜合應(yīng)用等溫吸附與解吸、等溫滴定量熱和衰減全反射紅外光譜技術(shù),測定了DNA與細(xì)胞-礦物復(fù)合物作用過程吸附量、親和力、熱力學(xué)參數(shù)及基團(tuán)變化,以揭示其與土壤組分的相互作用機(jī)制。并以有機(jī)磷農(nóng)藥降解質(zhì)粒為對象,結(jié)合熒光定量PCR和流式細(xì)胞術(shù)等方法,研究了游離態(tài)及固定態(tài)胞外DNA在環(huán)境中的存留時間,及向土著微生物進(jìn)行水平轉(zhuǎn)移的可能性。獲得如下主要結(jié)果:(1)揭示了DNA在土壤細(xì)菌-礦物復(fù)合物上的吸附行為及作用機(jī)制。選取鮭魚精DNA、兩種常見的土壤細(xì)菌(枯草芽孢桿菌和惡臭假單胞菌),以及三種典型土壤礦物(高嶺石、蒙脫石和針鐵礦)為實驗材料。表明所有細(xì)菌-礦物復(fù)合物對DNA的吸附可用Langmuir方程擬合。DNA在枯草芽孢桿菌-礦物復(fù)合物上的吸附量相比其在兩種單一組分上的等比例吸附值之和有所提高,表明細(xì)菌與礦物的吸附能增加該體系對DNA的吸附量。DNA與枯草芽孢桿菌-礦物復(fù)合物結(jié)合時的焓變主要來自細(xì)菌與DNA分子,其主要作用力為范德華力及氫鍵。DNA與惡臭假單胞菌-礦物復(fù)合物相互作用的情況則受到礦物類型的影響,其與惡臭假單胞菌-高嶺石復(fù)合物進(jìn)行吸附時主要作用力為范德華力及氫鍵;而與惡臭假單胞菌-蒙脫石/-針鐵礦的作用力則應(yīng)歸結(jié)于脫水作用及水分子重排引起的熵增。(2)成功構(gòu)建攜帶雙重標(biāo)記的有機(jī)磷農(nóng)藥降解質(zhì)粒,發(fā)現(xiàn)游離及固定態(tài)質(zhì)粒的轉(zhuǎn)化效率受質(zhì)粒類型、礦物種類及宿主類別等多因素影響。以有機(jī)磷高效降解質(zhì)粒pB17、廣宿主載體pBHR1及抗重金屬細(xì)菌惡臭假單胞菌X4的小質(zhì)粒為材料,對其進(jìn)行增強(qiáng)型綠色熒光蛋白基因egfp及卡那霉素抗性基因kmr雙重標(biāo)記,得到重組質(zhì)粒pRB17、pXr及pBr。并對三種游離態(tài)質(zhì)粒及與蒙脫石、高嶺石、針鐵礦吸附后的固定態(tài)質(zhì)粒進(jìn)行了實驗室水平的轉(zhuǎn)化實驗。結(jié)果表明,以廣宿主載體為基礎(chǔ)所構(gòu)建的降解質(zhì)粒具有向更多種類細(xì)菌轉(zhuǎn)移的能力。游離態(tài)的DNA較固定態(tài)DNA有更高的降解速率。DNA與礦物吸附后進(jìn)行轉(zhuǎn)化,所獲得轉(zhuǎn)化子的數(shù)量有不同程度的降低。游離態(tài)pBr轉(zhuǎn)化大腸桿菌DH5α其轉(zhuǎn)化效率可達(dá)2.98×105個轉(zhuǎn)化子/μg DNA,而與蒙脫石、高嶺石及針鐵礦吸附后,轉(zhuǎn)化效率分別下降了2~4個數(shù)量級。類似的結(jié)果也出現(xiàn)在pXr轉(zhuǎn)化DH5α的過程中,與三種礦物結(jié)合后的轉(zhuǎn)化效率下降了1~3個數(shù)量級。表明與DNA親和力強(qiáng)的礦物降低DNA轉(zhuǎn)化效率的作用更明顯。(3)闡明了游離及固定態(tài)質(zhì)粒在土壤環(huán)境中的穩(wěn)定性特征及生物可利用性。結(jié)果表明DNA在進(jìn)入土壤的最初階段降解迅速,3~7天后降解曲線逐漸平緩。游離態(tài)DNA較固定態(tài)DNA在土壤中的降解速度更快,經(jīng)DNA回收轉(zhuǎn)化大腸桿菌后,其轉(zhuǎn)化效率的下降也更為顯著。DNA被不同類型礦物吸附后其降解效率也存在差異。與高嶺石和針鐵礦相比,吸附于蒙脫石表面的DNA分子降解速率更慢,在第3天時殘留量仍保持在50%以上,而且第15天仍能成功轉(zhuǎn)化至大腸桿菌中。表明蒙脫石對DNA具有更好的保護(hù)作用,使其能免受核酸酶的降解,具有更長時間的生物可利用活性。(4)比較了多種檢測環(huán)境中小概率水平基因轉(zhuǎn)移事件的實驗手段,提出抗生素富集聯(lián)用流式細(xì)胞分選術(shù)能有效分離土壤中的罕見目標(biāo)細(xì)胞。證實了胞外DNA具有向土著微生物進(jìn)行轉(zhuǎn)移的能力。選擇游離態(tài)及蒙脫石固定態(tài)質(zhì)粒pBr及pXr作為土壤轉(zhuǎn)化實驗DNA供體,武漢市兩處地點的棕紅壤作為受體細(xì)菌來源,土樣樣品設(shè)置堆肥或有機(jī)磷農(nóng)藥處理組。利用熒光顯微鏡、標(biāo)記基因PCR擴(kuò)增及流式細(xì)胞術(shù)直接對土壤中的轉(zhuǎn)化子進(jìn)行檢測,檢測限均低于2×10-6,未能檢測到陽性轉(zhuǎn)化子的存在。通過抗生素富集及流式細(xì)胞多輪分選技術(shù)對樣品進(jìn)行篩選可有效將檢測限提高至10-11量級。所分得菌株均為來源于菜園土和添加農(nóng)藥處理實驗組的pBr質(zhì)粒轉(zhuǎn)化子,屬腸桿菌目腸桿菌科。廣宿主載體的使用、土壤微生物群落組成與結(jié)構(gòu)及農(nóng)藥施用對土壤菌群的選擇性壓力或為轉(zhuǎn)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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：X592

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