【作者】 李博；

【导师】 武俊；

【作者基本信息】 南京农业大学 ， 生态学， 2019， 硕士

【摘要】 β-甲氨基-L-丙氨酸(β-N-methylamino-L-alanine,BMAA)是蓝藻普遍产生的一种具有神经毒性的非蛋白质氨基酸,在自然界中存在游离态(Free BMAA)和蛋白结合态(Protein-associated BMAA)两种形式。研究发现,BMAA能够通过食物链进入西太平洋关岛生态系统和水生态系统,并沿食物链进行迁移放大,引发人类健康与生态安全风险。据报道,近年来我国包括鄱阳湖、太湖、巢湖在内的多个大型淡水湖泊都发生过大面积的蓝藻水华污染。考虑到蓝藻自身固氮的特性,在这些地区藻华会被打捞起来做成蓝藻有机肥,以实现藻华的无公害处理并达到有害物质的有效利用。施用蓝藻有机肥可能导致BMAA进入农田并在土壤与作物间进行迁移并富集在作物体内,而食用BMAA污染的农作物可能产生食品安全问题,从而危害人体健康,因此需要尽快开展农田土壤环境中BMAA毒素的调研工作。本文通过HPLC-MS/MS技术,首先在蓝藻有机肥中检测到了高含量的BMAA(1.8-16.3 μg/g),随后研究了两种形态BMAA在土壤和作物(小白菜)间的迁移累积过程。此外,研究报道BMAA会导致果蝇、蜜蜂和恒河猴出现运动神经功能紊乱和寿命缩短的症状。我们猜测BMAA可能会对土壤动物造成损伤,导致土壤动物的行为紊乱和寿命缩短,进而影响其生态功能,最终破坏土壤生态系统。本实验首先优化了样品中BMAA的提取方法。实验选择0.1M三氯乙酸(TCA)作为溶剂,通过超声细胞破碎和离心的方法提取出样品(蓝藻有机肥样品、作物各部位)中游离态BMAA,土壤样品无需进行超声破碎。通过盐酸高温水解提取出残余物中的蛋白结合态BMAA。上述提取液经过衍生化处理后,运用HPLC-MS/MS方法检测提取物中两种形态BMAA的含量。该方法简单、快速、准确、重复性好。平均加标回收率在80.51%到84.00%之间,平均标准偏差为2.42%到3.66%。作为实验的基础,我们在六种蓝藻有机肥中均检测到了两种形态的BMAA,其中游离态含量在1.8-16.3μg/g之间,蛋白结合态含量在3.43-13.67μg/g之间。检测结果说明在蓝藻堆肥过程中,由于自身非常稳定的化学性质,BMAA并不会被分解掉。通常认为BMAA在自然界中会优先以蛋白结合态存在,但有机肥中检测到的高含量游离态BMAA说明,堆肥的过程有可能促使蛋白结合态BMAA分解为游离态,进一步增加了 BMAA的生态风险。我们通过向土壤中一次性添加游离态BMAA溶液的方法进行作物吸收实验。考虑到在蓝藻有机肥中检测到的BMAA含量,将实验浓度设定为每克土壤中4μg BMAA。我们在土壤中种植小白菜,并在作物发芽后的第5天向土壤中一次性加入高浓度游离态BMAA溶液以达到实验浓度。在随后15天的试验周期中,分五次采集样品并植株分离,分别测定根、茎、叶以及土壤中BMAA含量,分析了 BMAA在作物体内的迁移累积情况。土壤中蛋白结合态BMAA含量从3.28 μg/g下降到0.97 μg/g,小白菜根、茎、叶中的BMAA含量随之增加,证明土壤中BMAA确实转移到了作物中。除了作物吸收实验,我们以模式生物秀丽隐杆线虫(Caenorhabditis elegans)作为试验对象,通过使线虫暴露在不同浓度(50、100、150μM)BMAA下,证明了 BMAA对线虫的寿命、繁殖、发育均产生了不利影响。线虫平均寿命随着BMAA浓度的提高分别缩短了 19%、36%和46%,并且产生了体长变短、繁殖提前、繁殖量减少的现象。我们通过转录组测序和RT-PCR实验,发现线虫的寿命相关基因表达发生了较大变化。由此证明,BMAA对秀丽隐杆线虫生态功能有明显负面影响。上述研究结果证明,BMAA可以在土壤与作物间发生迁移累积并最终富集在作物的可使用部位,并且通过使线虫暴露在不同浓度BMAA下,证明了BMAA对线虫的寿命、繁殖、发育均产生了不利影响。本研究结果为BMAA环境健康预警与土壤生态风险评价提供重要的科学依据。更多还原

【Abstract】 β-methylamino-L-alanine(BMAA)is a neurotoxic non-protein amino acid,which may be produced by all known groups of cyanobacteria,including cyanobacterial symbionts and free-living cyanobacteria.There are two forms of BMAA in nature,free BMAA and protein-associated BMAA.Ecology provides an important line of evidence supporting the occurrence of BMAA in the Western Pacific Guam ecosystem and many aquatic ecosystems combined with its possible transfer and bioaccumulation within the major food webs,some ending in human consumption,suggesting a potential for human health and ecological safety risks.Many of our large freshwater lakes(PoYanghu,Taihu,Chaohu)have presented serious cyanobacteria bloom pollution.In these areas,algal blooms are salvaged to make cyanobacteria composts due to its characteristic of nitrogen-fixation.This harmless treatment of algal blooms can make full use of hazardous.Fertilization of cyanobacterial composts in farmlands may also lead to BMAA contamination in soil and its possible transfer and bioaccumulation within major crops,thereby threatening human health.Therefore,research of BMAA toxin in water environment in China’s imminent.In this study,we first detected a high level of BMAA(1.8-16.3 μg g-1)in cyanobacterial composts by HPLC-MS/MS.Whereafter,we studied the transfer and bioaccumulation of two forms BMAA between soil and Chinese cabbage.As a neurotoxin,BMAA can cause functional disorder or decrease of life expectancy on Rhesus macaques,Drosophila melanogaster and honeybees.Thus,we speculated that this neurotoxin may have adverse effects on soil organisms.BMAA may cause injury to organism neurons and lead to the behavior disorders of soil organisms,and then influence their ecological functions,ultimately destroy the soil ecosystem.Firstly,we optimized the extraction method of BMAA in samples.0.1M trichloroacetic acid(TCA)was used as the solvent to extract free BMAA from the samples(cyanobacteria composts and parts of crops)by ultrasonication and centrifugation(ultrasionic cell-break was not needed for soil),subsequently,the protein-BMAA in residues will be extracted by hydrochloric acid hydrolysis.All of the samples were analyzed by HPLC-MS/MS to detect the concentration of two forms BMAA after derivatization.This method was simple,fast,accurate and well repeatability.The recovery of BMAA was from 80.51%to 84.00%and the relative SD was from 2.42%to 3.66%.As the basis of the assay we detected high levels of two forms BMAA in all six kinds of cyanobacterial composts with free BMAA ranged from 1.8-16.3μg/g and protein-associated ranged from 3.43-13.67μg/g.The results showed that BMAA could not be degraded during the composting due to the stable chemical properties.In particular,free BMAA will bind to protein after entering the organism and form a protein-associated state,which could be continuously accumulated and stored for a long time,but the high level of free BMAA we detected in composts demonstrated that protein-associated BMAA may degraded into free form during the composting process and this phenomenon may lead to ecological risk.Considering the high level of BMAA detected in cyanobacterial composts,on the 5th day of seedling stage,20 ml of high-concentration free BMAA solution was added into soil with the final concentration was 4 μg BMAA/g soil.In order to assess the health risks from cyanobacterial composts,we planted Chinese cabbage,a favorite vegetable in China,in BMAA-contaminated soil(4.0 μg BMAA/g soil)and detected the levels of free and protein-associated BMAA in soil and crop organs during the whole growth cycle by HPLC-MS/MS,respectively.Our results demonstrated that BMAA indeed transferred from soil to root,stem and leaf of Chinese cabbage during the growth cycle.The BMAA level finally accumulated in the edible portions was much higher than the initial level in soil,including 13.82 μg g-1 in leaf and 4.71 μg g-1 in stem.Besides,the concentration of protein-associated BMAA in soil declined from 3.28 μg/g to 0.97 μg/g.The model organism,Caenorhabditis elegans,was used as the researched object.We investigated the effects of BMAA on C.elegans at phenotypic,biochemical and molecular biological levels.Multiple endpoints,including lifespan,reproductive capacity and body size,as well as gene expression profiles,were determined after exposure of the nematodes to BMAA.As our expectation,BMAA shortened the lifespan and the body size.We observed BMAA hastened reproductive timing and caused the decrease of total number of offspring.The results of RNA-sequence and RT-PCR indicated that there were significant alterations of profile in a large number of genes.Thus,BMAA has negative effects on Caenorhabditis elegans.Our results demonstrated that BMAA can transfer between soil and crop and eventually enrich in the usable parts of the crop.We exposed Caenorhabditis elegans in BMAA and found BMAA had negative impact on the three indexes mentioned above,including decrease of lifespan,delayed growth and decrease of reproduction.Our results provide important scientific evidence for BMAA environmental health and soil ecological risk assessment.更多还原