大腸桿菌O157:H7對不同土壤礦物響應(yīng)的分子生物學(xué)機(jī)制
發(fā)布時間：2018-09-11 12:40
【摘要】：全球養(yǎng)殖業(yè)每年產(chǎn)生100~1000億噸的畜禽糞便,大部分畜禽糞便未經(jīng)有效處理直接作為肥料施入農(nóng)田,致使糞便中大量病原菌進(jìn)入土壤環(huán)境,對人類健康和食品安全構(gòu)成極大威脅。病原菌進(jìn)入土壤后,會吸附在土壤固相組分上,形成單個微菌落或生物被膜,從而在土壤中持久存在。因此,闡明病原菌對土壤礦物響應(yīng)的分子生物學(xué)機(jī)制,有助于深刻認(rèn)識土壤中病原菌的存活行為,同時對于控制及修復(fù)病原菌污染土壤具有重要理論和實(shí)際意義。本文選取了致病菌株大腸桿菌O157:H7為實(shí)驗菌株,研究了土壤常見礦物鐵氧化物(針鐵礦、赤鐵礦)、鋁氧化物(勃姆石)、層狀硅酸鹽礦物(高嶺石、蒙脫石、伊利石)對大腸桿菌O157:H7有關(guān)存活及毒性等基因表達(dá)的影響。主要結(jié)果有:1.不同土壤礦物對大腸桿菌O157:H7存活的影響平板計數(shù)結(jié)果顯示,與對照相比,在10 mmol/L KCl溶液中(p H=6.8),不同土壤礦物對大腸桿菌O157:H7的可培養(yǎng)數(shù)量沒有顯著影響,而結(jié)合熒光顯微鏡和LIVE/DEAD染色技術(shù),發(fā)現(xiàn)不同土壤礦物與大腸桿菌O157:H7結(jié)合后均可破壞大腸桿菌O157:H7細(xì)胞膜的完整性,從而導(dǎo)致細(xì)胞失活。胞內(nèi)活性氧(ROS)結(jié)果顯示,土壤礦物能誘導(dǎo)細(xì)菌胞內(nèi)ROS的產(chǎn)生,其中鐵鋁氧化物(針鐵礦、赤鐵礦、勃姆石)體系中,細(xì)菌胞內(nèi)ROS的含量是對照組的3.7~4.2倍;而層狀硅酸鹽(高嶺石、蒙脫石、伊利石)體系中,細(xì)菌胞內(nèi)ROS的含量僅為對照組的1.8~2.1倍,表明鐵鋁氧化物對胞內(nèi)活性氧的誘導(dǎo)作用強(qiáng)于層狀硅酸鹽礦物。2.不同土壤礦物對大腸桿菌O157:H7毒性、運(yùn)動等基因表達(dá)的影響大腸桿菌O157:H7的關(guān)鍵致病因子志賀毒素(Stx)有兩種主要形式:Stx1(stx1基因編碼)和Stx2(stx2基因編碼)。定量PCR結(jié)果顯示,土壤礦物可抑制大腸桿菌O157:H7的stx1、stx2基因表達(dá),而對細(xì)菌鞭毛調(diào)控基因flh D及編碼鞭毛蛋白的fli C基因的表達(dá)沒有顯著影響。土壤礦物可引起自由基清除酶基因sod B及與DNA修復(fù)相關(guān)的rec A基因的大量表達(dá),表明土壤礦物誘導(dǎo)的胞內(nèi)ROS對細(xì)菌DNA造成了氧化損傷。
[Abstract]:100 ~ 100 billion tons of livestock and poultry manure are produced every year in the global breeding industry. Most of the animal manure is directly applied as fertilizer to farmland without effective treatment, which results in a large number of pathogenic bacteria in feces entering the soil environment, which poses a great threat to human health and food safety. When the pathogen enters the soil, it will adsorb on the solid component of the soil and form a single microbacterium colony or biofilm, which will exist in the soil for a long time. Therefore, to elucidate the molecular biological mechanism of pathogen response to soil minerals is helpful to understand the survival behavior of pathogens in soil, and has important theoretical and practical significance in controlling and remediation of soil contaminated by pathogens. In this paper, the common mineral iron oxides (goethite, hematite), aluminum oxide (Bohmite), layered silicate minerals (kaolinite, montmorillonite) in soil were studied by using Escherichia coli O157:H7 as the experimental strain. The effect of Illite on the survival and toxicity of Escherichia coli O157:H7. The main result was 1: 1. The effects of different soil minerals on the survival of Escherichia coli O157:H7 showed that, compared with the control, in 10 mmol/L KCl solution (pH6. 8), different soil minerals had no significant effect on the culturable quantity of Escherichia coli O157:H7. Combined with fluorescence microscope and LIVE/DEAD staining, it was found that different soil minerals combined with Escherichia coli O157:H7 could destroy the integrity of Escherichia coli O157:H7 cell membrane, resulting in cell inactivation. The results of intracellular reactive oxygen species (ROS) showed that soil minerals could induce the production of intracellular ROS of bacteria, and the content of ROS in ferric aluminum oxide (goethite, hematite, Boehmite) system was 3.740. 2 times of that in control group. In the layered silicate (kaolinite, montmorillonite, Illite) system, the intracellular ROS content of bacteria was only 1.8 ~ 2. 1 times of that of the control group, indicating that the inductive effect of iron and aluminum oxide on intracellular reactive oxygen species was stronger than that of layered silicate mineral. 2. Effects of different soil Minerals on the expression of O157:H7 and exercise genes in Escherichia coli (Stx), the key pathogenic factor of E. coli O157:H7, (Stx) has two main forms: stx1 gene encoding (stx1 gene coding) and Stx2 (stx2 gene coding). The results of quantitative PCR showed that soil minerals could inhibit the expression of O157:H7 stx1,stx2 gene in E. coli, but had no significant effect on the expression of flh D gene and fli C gene encoding flagellin. Soil minerals could induce the expression of free radical scavenging enzyme gene sod B and rec A gene associated with DNA repair, indicating that intracellular ROS induced by soil minerals caused oxidative damage to bacterial DNA.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
本文編號：2236716
[Abstract]:100 ~ 100 billion tons of livestock and poultry manure are produced every year in the global breeding industry. Most of the animal manure is directly applied as fertilizer to farmland without effective treatment, which results in a large number of pathogenic bacteria in feces entering the soil environment, which poses a great threat to human health and food safety. When the pathogen enters the soil, it will adsorb on the solid component of the soil and form a single microbacterium colony or biofilm, which will exist in the soil for a long time. Therefore, to elucidate the molecular biological mechanism of pathogen response to soil minerals is helpful to understand the survival behavior of pathogens in soil, and has important theoretical and practical significance in controlling and remediation of soil contaminated by pathogens. In this paper, the common mineral iron oxides (goethite, hematite), aluminum oxide (Bohmite), layered silicate minerals (kaolinite, montmorillonite) in soil were studied by using Escherichia coli O157:H7 as the experimental strain. The effect of Illite on the survival and toxicity of Escherichia coli O157:H7. The main result was 1: 1. The effects of different soil minerals on the survival of Escherichia coli O157:H7 showed that, compared with the control, in 10 mmol/L KCl solution (pH6. 8), different soil minerals had no significant effect on the culturable quantity of Escherichia coli O157:H7. Combined with fluorescence microscope and LIVE/DEAD staining, it was found that different soil minerals combined with Escherichia coli O157:H7 could destroy the integrity of Escherichia coli O157:H7 cell membrane, resulting in cell inactivation. The results of intracellular reactive oxygen species (ROS) showed that soil minerals could induce the production of intracellular ROS of bacteria, and the content of ROS in ferric aluminum oxide (goethite, hematite, Boehmite) system was 3.740. 2 times of that in control group. In the layered silicate (kaolinite, montmorillonite, Illite) system, the intracellular ROS content of bacteria was only 1.8 ~ 2. 1 times of that of the control group, indicating that the inductive effect of iron and aluminum oxide on intracellular reactive oxygen species was stronger than that of layered silicate mineral. 2. Effects of different soil Minerals on the expression of O157:H7 and exercise genes in Escherichia coli (Stx), the key pathogenic factor of E. coli O157:H7, (Stx) has two main forms: stx1 gene encoding (stx1 gene coding) and Stx2 (stx2 gene coding). The results of quantitative PCR showed that soil minerals could inhibit the expression of O157:H7 stx1,stx2 gene in E. coli, but had no significant effect on the expression of flh D gene and fli C gene encoding flagellin. Soil minerals could induce the expression of free radical scavenging enzyme gene sod B and rec A gene associated with DNA repair, indicating that intracellular ROS induced by soil minerals caused oxidative damage to bacterial DNA.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61
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