环丙沙星（CIP）高、低累积菜心根际特性及其吸收累积影响机制

发布时间：2018-01-01 22:09

本文关键词：环丙沙星（CIP）高、低累积菜心根际特性及其吸收累积影响机制　出处：《暨南大学》2017年博士论文　论文类型：学位论文

【摘要】：抗生素在农业土壤、水体等环境介质中普遍被检出,直接影响到农产品安全,对环境和人体健康造成严重威胁。筛选对污染物低累积作物品种是在土壤普遍污染情况下确保农产品质量安全的重要策略。本文以人畜广泛使用且在环境介质中检出率和含量均较高的喹诺酮类环丙沙星(Ciprofloxacin,CIP)为目标化合物,以课题组前期筛选获的对CIP具有高累积菜心品种(Brassica parachinensis L.)四九菜心(SiJiu)和低累积菜心品种粗苔菜心(CuTai)为研究对象,通过研究两种菜心根系对CIP吸收动力学、吸收过程、亚细胞分布特征差异及两种菜心根系形态学、解剖学、细胞电生理学、根际微生态差异,揭示不同基因型菜心高、低累积CIP性状形成的生理学差异机制。本论文的主要研究结果如下:(1)高、低累积菜心根系对CIP吸收过程及吸收动力学差异采用裸根实验、分根实验和避光实验,考察了两种菜心根系对CIP的吸收过程。结果表明,两种菜心根系吸收CIP过程同时存在主动吸收和被动吸收,其中四九菜心和粗苔菜心根系对CIP主动吸收能力分别是被动吸收的2.39倍和1.71倍,说明两种菜心均以主动吸收为主,且四九菜心根系对CIP主动吸收过程是粗苔菜心的1.6倍,这可能是导致高累积形状形成的关键过程。两种菜心根系吸附和韧皮部作用相对于根系吸收作用非常小,但蒸腾作用显著促进两种菜心根系吸收CIP,是导致高累积吸收过程的重要因素。而且,四九菜心根系对CIP的吸收速率和根系细胞膜对CIP的亲和力即搬运能力均大于粗苔菜心,从而导致其根系更好地吸收和转运CIP,形成高累积特性。(2)高、低累积两种菜心根系形态学和解剖学差异通过根系扫描、番红固绿染色和透射电镜观察等技术,对两种菜心根系形态、根系组织结构和根系细胞超微结构进行观察。结果表明,不同浓度CIP均抑制了两种菜心根系生长和根系形态指标。两品种菜心相比,四九菜心的根表面积和根体积显著大于粗苔菜心,而根直径显著低于粗苔菜心,四九菜心根系输导组织中导管数变多,导管变粗等,有利于四九菜心对CIP的吸收和转运;而且CIP对四九菜心根尖细胞损伤较小,即使是在高浓度CIP胁迫下,其细胞结构仍然保持完整性且线粒体功能正常,从而保证高累积菜心品种长期、持续吸收与累积CIP。与四九菜心相比,CIP显著减少粗苔菜心根系木质部面积,使得CIP由根向地上部迁移作用减弱,而且CIP对粗苔菜心根尖细胞线粒体、细胞质的损伤较大,其中线粒体功能损伤会导致粗苔菜心根系主动吸收CIP供能不足。低累积菜心(粗苔)通过改变自身相关的组织结构和生理机制,以减少根系对CIP的吸收和转运以免遭毒害,属于“避性”抗污染胁迫机制。(3)高、低累积两种菜心根系CIP亚细胞分布及影响机制采用差速离心法对高、低累积菜心品种根系亚细胞中CIP的含量进行研究,发现CIP主要存储在根系的细胞器和细胞壁中,其次是细胞的可溶部分。在低和高浓度CIP处理下,粗苔菜心根细胞壁中CIP的分配比例(分别为40.6%和31.8%)均高于四九菜心(分别为36.5%和19.5%),即粗苔菜心根部细胞壁对CIP的滞留更加明显,减少了CIP向细胞内部转运和地上部累积。高污染水平下,细胞壁中CIP的分配比例均有所下降,而细胞器和细胞可溶部分CIP分配比例上升,且四九菜心细胞可溶部分中CIP分配比例低于粗苔菜心,说明液泡区室化对高累积品种根尖细胞中CIP的解毒起着重要作用。同时,两种浓度(1和10 mg/L)CIP处理下,四九菜心对CIP代谢率(分别为17.3%和22.0%)显著高于粗苔菜心(分别为3.70%和2.07%)。因此,高累积菜心(四九)根系具有较高代谢率和解毒能力,植株体内能够耐受较高浓度的CIP累积,属于“耐性”抗污染胁迫机制。通过油红染色实验研究发现,两种菜心根系组织中脂肪分布特征与亚细胞中CIP分布特征非常相似,说明菜心根系脂肪会影响亚细胞中CIP分布。通过共聚焦荧光显微镜对菜心根系吸收CIP过程进行观察,不仅直观地验证了两种菜心根系吸收CIP方式存在主动和被动两种方式,且通过荧光强度证实了四九菜心细胞壁和细胞质中CIP赋存量明显高于粗苔菜心。(4)高、低累积两种菜心根尖细胞电生理差异研究利用单电极微电极和非损伤微测技术,研究了CIP胁迫下两种菜心根系细胞电生理学差异。结果表明,在CIP胁迫下,两种菜心根尖细胞膜主要发生了去极化现象,且四九菜心根尖细胞膜电位的去极化水平大于粗苔菜心,根尖细胞膜电位的去极化大小决定了跨膜运动驱动力大小,说明CIP在四九菜心根系细胞膜中跨膜运动驱动力更大。通过对两种菜心根尖细胞H+进行监测,CIP处理下,均会导致两种菜心根尖细胞H+内流增加,其中粗苔根尖细胞H+内流依赖于CIP浓度,而四九仅在高浓度CIP处理下H+内流才会增加,初步推断CIP是以H+共转运的方式进入粗苔根尖细胞,H+内流增加是导致低累积菜心根系细胞损伤的直接原因。因此,细胞膜电势差可能是CIP在四九菜心跨膜运动的主要驱动力,而细胞膜两侧H+浓度可能是粗苔菜心的主要驱动力。(5)高、低累积两种菜心根际微生态差异研究通过根箱实验,研究了根际毫米级微域中环境因子、微生物和CIP消减行为之间的作用关系。研究表明,四九菜心土壤中CIP降解率高于粗苔菜心土壤,且在两种菜心土壤近根区尤其是2-4 mm处CIP的降解率最高。通过16S rDNA高通量测序,发现高累积品种菜心土壤中微生物丰富度和多样性均大于低累积品种,尤其是近根区和根区土壤中二者差异更显著,且含CIP特异降解菌功能的变形菌门明显高于粗苔,这可能是造成四九根际去除CIP能力更强和体内吸收累积CIP更多的重要原因之一。相关性分析显示,低浓度CIP处理下,土壤中微生物多样性促进了土壤中CIP消减,而高浓度下,土壤中微生物多样性受到抑制,从而降低了两种菜心近根区和远根区土壤中CIP消减。同时,两种菜心根际微域土壤中水溶性有机碳(DOM)含量与微生物多样性相关性不同,是导致两种菜心根际微域中CIP消减的主要原因之一。
[Abstract]:Antibiotics in agricultural soil, water environment is generally positive in the medium, directly affects the safety of agricultural products, which poses a serious threat to the environment and human health. Screening of pollutants in low cumulative crop varieties is an important strategy to ensure the quality and safety of agricultural products in the case of contaminated soil commonly. The people and livestock are widely used and the detection rate and content in the medium with higher ciprofloxacin (Ciprofloxacin, CIP) as the target compound in ourprevious were on CIP with high cumulative (Brassica parachinensis L.) Chinese cabbage cabbage 49 (SiJiu) and low accumulation of Chinese cabbage cabbage thick moss (CuTai) as the research object, through the research of two kinds of cabbage root CIP uptake kinetics, absorption process, differences in subcellular distribution characteristics and two kinds of cabbage root morphological, anatomical, electrophysiological, rhizosphere micro ecological differences, reveal the different Genotypes of brassicaparachinensis high, differences in the physiological mechanism to form a low accumulation of CIP traits. The main results are as follows: (1) high and low accumulation of Brassica chinensis root on CIP absorption process and absorption kinetics of the difference with bare root experiment, split root experiment and light experiments, investigated the absorption process of two vegetables heart roots on CIP two. The results show that the absorption process of CIP cabbage root and passive and active uptake, including 49 Chinese cabbage and coarse fur brassicaparachinensis root active absorption capacity of CIP are passively absorbed 2.39 times and 1.71 times, that two are in active absorption of cabbage, cabbage and 49 root of CIP active absorption process is 1.6 times as wide moss cabbage heart. This may be the cause of the key process of high cumulative shape. Two kinds of cabbage root adsorption and phloem effect is very small relative to the root resorption, but transpiration significantly promoted the two kinds of Chinese Cabbage The root absorption of CIP, is an important factor leading to high cumulative absorption process. Moreover, the affinity of the 49 cabbage roots on CIP absorption rate and root cell membrane on CIP removal were thick moss flowering Chinese cabbage, which lead to better root uptake and translocation of CIP, the formation of high accumulation characteristics. (2) high and low accumulation two kinds of morphological and anatomical differences by brassicaparachinensis root root scanning, safranin fast green staining and transmission electron microscope technique, two kinds of cabbage root morphology, root tissue structure and root cell ultrastructure were observed. The results showed that different concentrations of CIP was inhibited by two kinds of cabbage root growth and root morphology index. Compared with two varieties 49 Chinese cabbage, cabbage root surface area and root volume was significantly greater than that of coarse fur cabbage, root diameter was significantly lower than that of thick moss cabbage, 49 catheter brassicaparachinensis root conducting tissue number, duct thickening, favorable In 49 Chinese cabbage absorption and translocation of CIP in CIP of 49; and the damage of root tip cells of cabbage is small, even at high concentrations of CIP, the cell structure is still intact and normal mitochondrial function, so as to ensure the long-term sustained high accumulation of Chinese cabbage, the absorption and accumulation of CIP. and CIP decreased significantly compared to 49 Chinese cabbage, thick fur cabbage root xylem area, the CIP from root to shoot migration effect is reduced, and the mitochondrial CIP thick moss cabbage root tip cells, cytoplasm damage large, which will lead to the damage of mitochondrial function of coarse root active absorption Taicai heart CIP for energy shortage. The low accumulation of cabbage (thick moss) by changing their relevant organizational structure and the physiological mechanism, to reduce the absorption and transport of CIP from roots to poison, belong to "avoid" anti pollution stress mechanism. (3) high and low cumulative two cabbage root CIP subcellular distribution and The influence mechanism of high speed centrifugation, research content of root subcellular low cumulative flowering Chinese cabbage varieties of CIP, CIP was mainly stored in the root cell organelles and cell wall, followed by the soluble fraction of cells. At low and high concentration of CIP, proportion of coarse fur cabbage in root cell wall CIP (40.6% and 31.8%) were higher than that of 49 Chinese Cabbage (36.5% and 19.5%), which is a coarse fur cabbage root cell wall retention on CIP is more obvious, reduced CIP to intracellular transport and shoot accumulation. High levels of pollution, the distribution proportion of CIP in the cell wall decreased, and cell organelles the soluble fraction of CIP and cell proportion increased, and the proportion of CIP cells in the soluble part of distribution of 49 Chinese cabbage below the coarse fur that vacuolar compartmentalization of Chinese cabbage, which plays an important role in the high accumulation of varieties in root tip cells of CIP detoxification. At the same time, two different concentrations (1 and 1 0 CIP, 49 mg/L) on cabbage CIP metabolic rate (17.3% and 22%) was significantly higher than that of coarse fur cabbage (3.70% and 2.07%). Therefore, the high accumulation of cabbage (49) root has a high metabolic rate and detoxification capacity, plants can tolerate high concentrations of CIP accumulation, which belongs to the "tolerance" anti pollution stress mechanism. Through the experimental study of oil red staining, characteristics and subcellular distribution characteristics of CIP is very similar to fat distribution of two kinds of cabbage root tissue, indicating brassicaparachinensis root fat will affect the subcellular distribution of CIP was observed by CIP. The process of cabbage root absorption confocal fluorescence microscopy, not only verified the root of two with active and passive absorption of CIP exists in two ways, and the fluorescence intensity confirmed that 49 Chinese cabbage cell wall and cytoplasm CIP Fu stock was significantly higher than that of coarse fur heart. (4) high and low cumulative two Vegetables heart root tip cells electrophysiological differences study using single electrode microelectrode and non-invasive micro-test technique under CIP stress were studied in two kinds of Chinese cabbage root cell electrophysiological differences. The results showed that under CIP stress, two kinds of cabbage root tip cells occurred mainly to membrane polarization and membrane potential to root tip cells of 49 Chinese cabbage the polarization level is greater than the coarse fur cabbage, the apical cell membrane potential depolarization determines the size of the driving force of the transmembrane movement, CIP in 49 Chinese cabbage root cell membrane in the transmembrane movement of a greater driving force. Through the monitoring of the two kinds of cabbage root tip cells of H+, CIP, will lead to increased two kinds of cabbage root the H+ cells in the flow, and the coarse fur of root tip cells in the inflow of H+ is dependent on the concentration of CIP, while the 49 only in the high concentration of CIP will increase the flow of H+, it is concluded that CIP is entering the coarse root tip cells with H+ moss co transport way, H+ In the current increase is a direct cause of low cumulative brassicaparachinensis root cell damage. Therefore, the cell membrane potential may be the main driving force of the movement in the 49 transmembrane CIP of flowering Chinese cabbage, and both sides of the membrane H+ concentration may be the main driving force of coarse fur. Cabbage (5) high and low cumulative two vegetable heart rhizosphere micro ecology the difference of rhizobox experiment, studied the rhizosphere environment of micro domain millimeter, microbial and CIP subduction behavior relationships. The study shows that the CIP degradation rate was 49 higher than that of the coarse moss soil soil in cabbage cabbage, and cabbage in two kinds of soil near the root zone especially the degradation of 2-4 mm CIP was the highest by 16S rDNA high-throughput sequencing, and found that high accumulation of microbial richness and diversity of soil cabbage varieties were higher than low accumulation varieties, especially the two district near the root and root zone soil more significant differences, and containing CIP specific degrading bacteria to change The door shaped bacteria was significantly higher than that of coarse fur, which is probably caused by the 49 rhizosphere CIP removal ability and in vivo absorption and accumulation of one of the important reasons for CIP more. Correlation analysis showed that the low concentration of CIP treatment, the soil microbial diversity in soil promoted the reduction of CIP, while high concentration, microbial diversity in soil was inhibited two, to reduce the soil near the root zone and cabbage root zone in CIP far reduction. At the same time, two kinds of cabbage rhizosphere soil water soluble organic carbon (DOM) content between different microbial diversity, is a major cause of CIP reduction of two Chinese cabbage Rhizosphere.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X173;S634.5

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