本文關(guān)鍵詞：環(huán)丙沙星（CIP）高、低累積菜心根際特性及其吸收累積影響機(jī)制　出處：《暨南大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 菜心 抗生素 吸收累積差異 生理生化特征 亞細(xì)胞分布 電生理 根際效應(yīng)

【摘要】：抗生素在農(nóng)業(yè)土壤、水體等環(huán)境介質(zhì)中普遍被檢出,直接影響到農(nóng)產(chǎn)品安全,對環(huán)境和人體健康造成嚴(yán)重威脅。篩選對污染物低累積作物品種是在土壤普遍污染情況下確保農(nóng)產(chǎn)品質(zhì)量安全的重要策略。本文以人畜廣泛使用且在環(huán)境介質(zhì)中檢出率和含量均較高的喹諾酮類環(huán)丙沙星(Ciprofloxacin,CIP)為目標(biāo)化合物,以課題組前期篩選獲的對CIP具有高累積菜心品種(Brassica parachinensis L.)四九菜心(SiJiu)和低累積菜心品種粗苔菜心(CuTai)為研究對象,通過研究兩種菜心根系對CIP吸收動(dòng)力學(xué)、吸收過程、亞細(xì)胞分布特征差異及兩種菜心根系形態(tài)學(xué)、解剖學(xué)、細(xì)胞電生理學(xué)、根際微生態(tài)差異,揭示不同基因型菜心高、低累積CIP性狀形成的生理學(xué)差異機(jī)制。本論文的主要研究結(jié)果如下:(1)高、低累積菜心根系對CIP吸收過程及吸收動(dòng)力學(xué)差異采用裸根實(shí)驗(yàn)、分根實(shí)驗(yàn)和避光實(shí)驗(yàn),考察了兩種菜心根系對CIP的吸收過程。結(jié)果表明,兩種菜心根系吸收CIP過程同時(shí)存在主動(dòng)吸收和被動(dòng)吸收,其中四九菜心和粗苔菜心根系對CIP主動(dòng)吸收能力分別是被動(dòng)吸收的2.39倍和1.71倍,說明兩種菜心均以主動(dòng)吸收為主,且四九菜心根系對CIP主動(dòng)吸收過程是粗苔菜心的1.6倍,這可能是導(dǎo)致高累積形狀形成的關(guān)鍵過程。兩種菜心根系吸附和韌皮部作用相對于根系吸收作用非常小,但蒸騰作用顯著促進(jìn)兩種菜心根系吸收CIP,是導(dǎo)致高累積吸收過程的重要因素。而且,四九菜心根系對CIP的吸收速率和根系細(xì)胞膜對CIP的親和力即搬運(yùn)能力均大于粗苔菜心,從而導(dǎo)致其根系更好地吸收和轉(zhuǎn)運(yùn)CIP,形成高累積特性。(2)高、低累積兩種菜心根系形態(tài)學(xué)和解剖學(xué)差異通過根系掃描、番紅固綠染色和透射電鏡觀察等技術(shù),對兩種菜心根系形態(tài)、根系組織結(jié)構(gòu)和根系細(xì)胞超微結(jié)構(gòu)進(jìn)行觀察。結(jié)果表明,不同濃度CIP均抑制了兩種菜心根系生長和根系形態(tài)指標(biāo)。兩品種菜心相比,四九菜心的根表面積和根體積顯著大于粗苔菜心,而根直徑顯著低于粗苔菜心,四九菜心根系輸導(dǎo)組織中導(dǎo)管數(shù)變多,導(dǎo)管變粗等,有利于四九菜心對CIP的吸收和轉(zhuǎn)運(yùn);而且CIP對四九菜心根尖細(xì)胞損傷較小,即使是在高濃度CIP脅迫下,其細(xì)胞結(jié)構(gòu)仍然保持完整性且線粒體功能正常,從而保證高累積菜心品種長期、持續(xù)吸收與累積CIP。與四九菜心相比,CIP顯著減少粗苔菜心根系木質(zhì)部面積,使得CIP由根向地上部遷移作用減弱,而且CIP對粗苔菜心根尖細(xì)胞線粒體、細(xì)胞質(zhì)的損傷較大,其中線粒體功能損傷會(huì)導(dǎo)致粗苔菜心根系主動(dòng)吸收CIP供能不足。低累積菜心(粗苔)通過改變自身相關(guān)的組織結(jié)構(gòu)和生理機(jī)制,以減少根系對CIP的吸收和轉(zhuǎn)運(yùn)以免遭毒害,屬于“避性”抗污染脅迫機(jī)制。(3)高、低累積兩種菜心根系CIP亞細(xì)胞分布及影響機(jī)制采用差速離心法對高、低累積菜心品種根系亞細(xì)胞中CIP的含量進(jìn)行研究,發(fā)現(xiàn)CIP主要存儲(chǔ)在根系的細(xì)胞器和細(xì)胞壁中,其次是細(xì)胞的可溶部分。在低和高濃度CIP處理下,粗苔菜心根細(xì)胞壁中CIP的分配比例(分別為40.6%和31.8%)均高于四九菜心(分別為36.5%和19.5%),即粗苔菜心根部細(xì)胞壁對CIP的滯留更加明顯,減少了CIP向細(xì)胞內(nèi)部轉(zhuǎn)運(yùn)和地上部累積。高污染水平下,細(xì)胞壁中CIP的分配比例均有所下降,而細(xì)胞器和細(xì)胞可溶部分CIP分配比例上升,且四九菜心細(xì)胞可溶部分中CIP分配比例低于粗苔菜心,說明液泡區(qū)室化對高累積品種根尖細(xì)胞中CIP的解毒起著重要作用。同時(shí),兩種濃度(1和10 mg/L)CIP處理下,四九菜心對CIP代謝率(分別為17.3%和22.0%)顯著高于粗苔菜心(分別為3.70%和2.07%)。因此,高累積菜心(四九)根系具有較高代謝率和解毒能力,植株體內(nèi)能夠耐受較高濃度的CIP累積,屬于“耐性”抗污染脅迫機(jī)制。通過油紅染色實(shí)驗(yàn)研究發(fā)現(xiàn),兩種菜心根系組織中脂肪分布特征與亞細(xì)胞中CIP分布特征非常相似,說明菜心根系脂肪會(huì)影響亞細(xì)胞中CIP分布。通過共聚焦熒光顯微鏡對菜心根系吸收CIP過程進(jìn)行觀察,不僅直觀地驗(yàn)證了兩種菜心根系吸收CIP方式存在主動(dòng)和被動(dòng)兩種方式,且通過熒光強(qiáng)度證實(shí)了四九菜心細(xì)胞壁和細(xì)胞質(zhì)中CIP賦存量明顯高于粗苔菜心。(4)高、低累積兩種菜心根尖細(xì)胞電生理差異研究利用單電極微電極和非損傷微測技術(shù),研究了CIP脅迫下兩種菜心根系細(xì)胞電生理學(xué)差異。結(jié)果表明,在CIP脅迫下,兩種菜心根尖細(xì)胞膜主要發(fā)生了去極化現(xiàn)象,且四九菜心根尖細(xì)胞膜電位的去極化水平大于粗苔菜心,根尖細(xì)胞膜電位的去極化大小決定了跨膜運(yùn)動(dòng)驅(qū)動(dòng)力大小,說明CIP在四九菜心根系細(xì)胞膜中跨膜運(yùn)動(dòng)驅(qū)動(dòng)力更大。通過對兩種菜心根尖細(xì)胞H+進(jìn)行監(jiān)測,CIP處理下,均會(huì)導(dǎo)致兩種菜心根尖細(xì)胞H+內(nèi)流增加,其中粗苔根尖細(xì)胞H+內(nèi)流依賴于CIP濃度,而四九僅在高濃度CIP處理下H+內(nèi)流才會(huì)增加,初步推斷CIP是以H+共轉(zhuǎn)運(yùn)的方式進(jìn)入粗苔根尖細(xì)胞,H+內(nèi)流增加是導(dǎo)致低累積菜心根系細(xì)胞損傷的直接原因。因此,細(xì)胞膜電勢差可能是CIP在四九菜心跨膜運(yùn)動(dòng)的主要驅(qū)動(dòng)力,而細(xì)胞膜兩側(cè)H+濃度可能是粗苔菜心的主要驅(qū)動(dòng)力。(5)高、低累積兩種菜心根際微生態(tài)差異研究通過根箱實(shí)驗(yàn),研究了根際毫米級微域中環(huán)境因子、微生物和CIP消減行為之間的作用關(guān)系。研究表明,四九菜心土壤中CIP降解率高于粗苔菜心土壤,且在兩種菜心土壤近根區(qū)尤其是2-4 mm處CIP的降解率最高。通過16S rDNA高通量測序,發(fā)現(xiàn)高累積品種菜心土壤中微生物豐富度和多樣性均大于低累積品種,尤其是近根區(qū)和根區(qū)土壤中二者差異更顯著,且含CIP特異降解菌功能的變形菌門明顯高于粗苔,這可能是造成四九根際去除CIP能力更強(qiáng)和體內(nèi)吸收累積CIP更多的重要原因之一。相關(guān)性分析顯示,低濃度CIP處理下,土壤中微生物多樣性促進(jìn)了土壤中CIP消減,而高濃度下,土壤中微生物多樣性受到抑制,從而降低了兩種菜心近根區(qū)和遠(yuǎn)根區(qū)土壤中CIP消減。同時(shí),兩種菜心根際微域土壤中水溶性有機(jī)碳(DOM)含量與微生物多樣性相關(guān)性不同,是導(dǎo)致兩種菜心根際微域中CIP消減的主要原因之一。
[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.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：X173;S634.5

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