【作者】 王建升；

【导师】 汪俏梅；

【作者基本信息】 浙江大学 ， 蔬菜学， 2009， 博士

【摘要】 腐马素（fumonisins）是由串珠镰刀菌和再誉镰刀菌等镰刀菌产生的一类真菌毒素。由于其化学结构与动植物中的神经鞘氨醇类物质类似，可以引起动物各种疾病以及植物病害。一方面，腐马素对某些牲畜有急性毒性及潜在的致癌性，如引起马脑炎、猪肺水肿或大鼠肝癌，还能引起人类食道癌和神经管缺陷，由于该毒素污染广泛，在玉米、玉米制品以及芦笋等农产品中都有报道，严重威胁食品安全及人类和动物的健康；另一方面，腐马素可以诱导植物的细胞程序化死亡以及过敏性反应，在模式植物拟南芥中腐马素诱导细胞程序化死亡是研究植物对化学胁迫的抗性、植物免疫机理以及神经鞘脂功能的良好体系。本研究中，建立了用于玉米等食品中腐马素检测的HPLC-ELSD方法，分析了我国不同地区，特别是食管癌高发区玉米和玉米制品上腐马素污染状况；结合LC-MS分析了浙江省芦笋中腐马素污染情况；并利用PCR检测和产毒培养等方法，研究了玉米及芦笋中产生腐马素的原因。此外，以拟南芥为材料，筛选并初步分析了两个抗FB₁的突变体，同时研究了芥子油苷在拟南芥对FB₁抗性中的作用。主要研究结果如下：利用腐马素标准品和野生型串珠镰刀菌A0149产毒培养玉米为材料，结合常规的HPLC-OPA衍生法建立了不需要衍生的快速可靠的HPLC-ELSD方法，腐马素B1（FB₁）在3-24μg之间其物质的量与峰面积分别取自然对数后有良好的线性关系，最低检出限约为60 ng，可以同时检测B系列四种腐马素。利用该方法分析了我国东北地区、中东部地区和东南部地区玉米中腐马素的污染水平，结果表明，腐马素的整体污染水平较低，只检出了FB₁，中东地区玉米阳性样品中腐马素范围在0．25-1．8μg/g（平均，0．74μg/g），检出率为66．7%（15/24），东北地区玉米阳性样品中腐马素范围在0．21-0．29μg/g（平均，0．24μg/g），检出率为28．6%（6/21），东南部地区玉米阳性样品中腐马素范围在0．30-3．13μg/g（平均，0．47μg/g），检出率为30．0%（6/20）。我国的河南林县被认为是世界上食道癌发生与玉米中腐马素污染密切相关的地区之一。我们利用建立的HPLC-ELSD方法，研究了该地区不同来源和不同表观特征的玉米，以及不同玉米制品中腐马素的污染状况。结果表明，来自农户、粮仓，中心市场，商店以及超市的共140份玉米样品中FB₁检出率分别为61．5%（16/26），50%（12/24），33．3%（12/36），17%（2/12），O%（0/6）。来自粮库的玉米样品中腐马素污染水平最高（0．25-1．80μg/g；平均，0．73μg/g），其次是来自农户（0．25-1．80μ/g；平均，0．73μg/g），而来自中心市场（0．25-1．10μg/g；平均，0．51μg/g）和商店较轻（0．22-0．34μg/g；平均，0．28μg/g）。对80个来自当地农户玉米样品分析后发现，24个霉变玉米样品中18个检出了较高水平的腐马素B1（0．28-3．30μg/g；平均，1．58μg/g），而56份外观健康的玉米中有20份也检出了腐马素B1（0．21-0．82μg/g；平均，0．46μg/g）。来自中心市场的11 5份玉米食品和饲料样品中，饲料中腐马素的含量最高（0．30-3．13μg/g；平均，1．50μg/g），其次是非加工食品（0．31-0．63μg/g，平均，0．47μg/g），加工食品中污染最轻（0．21-0．28μg/g，平均，0．25μg/g），其检出率分别为53．6%，33．3%和1 7．9%。总之，来自林县地区的玉米食品和玉米饲料中腐马素污染水平较低（＜2μg/g），但是对粮仓和农户贮藏玉米的腐马素污染还需要采取措施加以控制。为了研究玉米中腐马素污染的原因，我们采用形态学鉴定和分子检测相结合的办法，研究了收集玉米中镰刀菌的污染状况，并通过产毒培养方法分析了分离菌株的产毒能力。总体看来，玉米样品中真菌，镰刀菌和腐马素产毒菌的污染率分别为42．1 8%，35．94%和29．69%，其中镰刀菌和产毒菌分别占总污染菌的74．1 9%（23/31）和61．29%（19/31）。PCR检测和产毒培养结果表明，共有三种镰刀菌在玉米培养基上产生了腐马素，分别为串珠镰刀菌、再誉镰刀菌和胶孢镰刀菌。产毒能力分析表明，不同菌种之间以及相同菌种的不同菌株之间产毒能力差异显著，产毒菌株的产毒能力从1 86μg/g到16784μg/g，平均为4637μg/g，其中FB₁占主要部分为87．1%（75．8-94．6%，平均产量4041μg/g）；第二类主要毒素为FB₂，平均产量为642μg/g，范围为46-2308μg/g，而且所有的串珠镰刀菌和再誉镰刀菌也都产生FB₃，平均产量为1 89μg/g，范围为42-711μg/g。不同地区腐马素产毒菌株污染状况不同，其中以中东部地区污染最重，共从24份样品中分离到14个污染菌，其中9个为产毒菌，而且污染的镰刀菌种类也最多，除了串珠镰刀菌外，还有再誉镰刀菌、禾谷镰刀菌和木贼镰刀菌；东南部地区则最轻，从19份样品中分离到5个菌株，而且只有一株产毒菌株；东北地区21份玉米中共分离到12个菌株，所有的9个镰刀菌均为产毒菌株；包括两个产毒的胶孢镰刀菌。我们的研究表明，这些外观洁净的食用玉米中仍然存在腐马素及产毒菌的污染，这可能是采前受到产毒病原菌侵染，但采后过程中可能不适合产毒造成的，由于这些产毒菌的产毒能力较强，如果条件适宜，仍然存在大量毒素污染的可能性。HPLC-ELSD和LC-MS分析都表明浙江省地区食用芦笋样品中没有腐马素污染。从22份芦笋样品中共分离到9株再誉镰刀菌，5株尖孢镰刀菌，1株木贼镰刀菌和1株锐顶镰刀菌，复合PCR分析表明，所有的再誉镰刀菌均扩增出了腐马素生物合成基因FUM1和FUM8片段，而且其产毒能力较强，在玉米培养基中总腐马素的平均产量为1603．5μg/g，范围27．8-4204．0μg/g，产生FB₁为148．2-3850．2μg/g（平均，1355．9μg/g），产生FB2为21．4-543．0μg/g（平均，1 53．1μg/g），产生FB₃为0-434．2μg/g（平均，106．4μg/g），其中5株属于高产毒菌（＞500μg/g），3株属于中产毒菌（50-500μg/g）。而其他菌株则只检测到镰刀菌特异的ITS片段，产毒培养和液相分析证实了存在产毒争议的尖孢镰刀菌确实不能产生FBs。我们的结果表明，浙江省食用芦笋中没有腐马素的污染，但能够产生真菌毒素的镰刀菌污染较重，这些菌株不仅可以引起芦笋的病害而且可以产生对人体有害的真菌毒素，应引起重视并加以控制。本研究还建立了一种拟南芥抗腐马素突变体的筛选方法，利用1μM的FB₁培养基从EMS诱变的哥伦比亚生态型Col-0突变体库中筛选得到两株抗FB₁的突变体fbr41和fbr42。在正常条件下，两个突变体表现出植株矮化，叶片边缘锯齿，以及侧根相对发达等异常表型；在1μM FB₁培养基上，根毛发达，叶片变硬，只在根等部位产生少量活性氧，其抗性远强于激素突变体jar1，ein3-1，npr1和转基因NahG，最大FB₁抗性浓度在2μM左右；遗传学分析表明，fbr41为隐性单基因突变。10μM FB₁外源处理野生型拟南芥Col-0后，HPLC分析表明，短链脂肪类芥子油苷含量下降，而长链脂肪类芥子油苷含量略有升高；吲哚基-3-甲基芥子油苷（Indol-3-ylmethyl GS，IM）的含量明显下降，和而4-甲氧吲哚基-3-甲基芥子油苷（4-methoxyindol-3-ylmethyl GS，4IM）和1-甲氧吲哚基-3-甲基芥子油苷（1-methoxyindol-3-ylmethyl GS，1IM）则明显升高；而长链脂肪类芥子油苷缺失但吲哚类含量高的突变体gsm1-3和所有芥子油苷组分都低的突变体gcc8同样在FB₁处理后被极大地诱导了4IM和1IM，而且gsm1-3表现出稍好的抗性，这预示不是长链脂肪类芥子油苷而可能是4IM和1IM参与了对FB₁的抗性作用。对芥子油苷生物合成途径相关基因分析表明，与抗病相关的camalexin和IAA生物合成基因（PAD3和NIT2）的表达在处理与对照之间没有明显差异，而参与吲哚类芥子油苷生物合成的CYP83B1却极大的上调；此外，我们对筛选的两个拟南芥抗FB₁突变体的芥子油苷及其生物合成相关基因也进行了分析，结果表明，两个突变体中均含有显著高于野生型的4IM和1IM含量，而且CYP83B1也被较早诱导。这些结果表明，拟南芥中吲哚类芥子油苷4IM和1IM的合成可能与拟南芥对FB₁抗性有关，但其具体作用机理尚待进一步研究来阐明。更多还原

【Abstract】 Fumonisins （FB） are a group of mycotoxins produced by Fusarium spp.includingFusarium verticillioides and Fusarium proliferatum.Due to the structural similaritywith sphingolipids,fumonisins can cause diseases in animals and plants.Fumonsinshave been demonstrated to cause several fatal diseases in animals,such asleukoencephalomalacia in horses,pulmonary oedema and hepatic cancer in rats.Inhumans,fumonisins are associated with oesophageal cancer and birth neural tubedefects.Fumonisin contamination occurs ubiquitously-it was found in manyagricultural products,such as corn,corn products and asparagus spears,which isassociated with food safety,livestock loss and human health risks.Fumonisin B1 （FB₁）induces programmed cell death （PCD） and hypersensitive reaction （HR） in plant,providing a good system to study innate immune response,chemical stress andsphinglipids function in Arabidopsis thaliana （Arabidopsis）.In present study,aneffective high-performance liquid chromatography coupled with an evaporative laserscattering detector （HPLC-ELSD） method was developed for detection of fumonisinsin food products.The method was used for the analysis of fumonisins contamination incorn products from different areas including a high-risk area for esophageal cancer.The fumonisins level was also assessed by LC-MS detection in asparagus spears fromZhejiang Province.Using PCR and fumonisin-producing culture analysis,the factorscausing fumonisin production in corn and asparagus spears were also discussed.Inaddition,two Arabidopsis FB₁-resistant mutants were isolated,and the role ofglucosinolates in FB₁-induced PCD was studied in Arabidopsis.Following are themain results.A new HPLC-ELSD method was developped for detection and quantification offumonisins in foods without any prior derivatization of the samples,it is simple andfast comparing to the conventional OPA derivatization method.We analyzed thefumonisins in corn-based food samples from central markets in eastern China byHPLC-ELSD method.The results showed that FB₁ was the main contaminant in thesamples.The overall level of fumonisin contamination was relatively low,with a range of 0.25 tol.80μg/g （mean 0.74μg/g） in 66.7% （16 of 24） of corn samples fromMiddle-eastern Area,0.21 to 0.29μg/g （mean 0.24μg/g） in 28.6% （6 of 21） of cornsamples from Northeastern Area,and 0.30 to 3.13μg/g （mean 0.47μg/g） in 30.0% （6of 20） of corn samples from Southeastern Area.The level of mycotoxin fumonisins in corn-based food and feed collected fromLinxian County,a high-risk area for esophageal cancer in China,was analyzed byHPLC-ELSD.A total of 104 corn kernel samples was obtained from local household,granary,wholesale market （central market）,and retail markets （store and supermarket）.Fumonisin B1 was detected in the samples from household,granary,central market,and store,with a positive rate of 61.5%,50%,33.3%,and 17%,respectively.Nofumonisin was detected in samples from supermarket.The highest FB₁ levels （0.30 to3.20μg/g;mean,1.42μg/g） were found in samples from granary,followed byhousehold （0.25 to 1.80μg/g;mean,0.73μg/g）,central market （0.25 to 1.10μg/g;mean,0.51μg/g）,and store （0.22 to 0.34μg/g;mean,0.28μg/g）.Among the 80 cornkernel samples collected from local household,18 of 24 （75.0%） moldy samplescontained high levels of FB₁ （0.28 to 3.30μg/g;mean,1.58μg/g）,and 20 of 56（35.7%） apparently healthy samples contained low levels of FB₁ （0.21 to 0.82μg/g;mean,0.46μg/g）.As central market plays an important role in trade of corn-basedfood and feed in China,a total of 115 corn-based food and feed samples were collectedfrom local central market.The highest FB1 levels （0.30 to 3.13μg/g;mean,1.50μg/g）were found in feed,followed by unprocessed food （0.31 to 0.63μg/g;mean,0.47μg/g）and processed food （0.21 to 0.28μg/g;mean,0.25μg/g）.The positive incidence of FB₁in feed,unprocessed,and processed food were 53.6%,33.3% and 17.9%,respectively.In conclusion,the results show that corn-based food and feed from Linxian Countycontained low level of FB₁ （＜2μg/g） in general,but efforts should be made to controlthe fumonisin contamination in corn kernels stored in granary and household.In the survey to elucidate the relationship between fumonisin-producing fungi andfumonisin contamination in corn,morphology characterization and PCR detectionwere conducted to identifying Fusaiusm spp.Isolates,whose capacity of producingfumonisins were then analyzed by inoculating cracked maize kernels （CMK） medium. Generally,the contamination rate of fungi,Fusarium spp.and fumonisin-producingfungi is 42.18%,35.94% and 29.69%,respectively,and the proportion of Fusariumand fumonisin-producing fungi in all contamination isolates is 74.19% （23/31） and61.29% （19/31）,respectively.Our results showed that three Fusarium speciesincluding F.versillioides,F.proliferatum and F.subglutinans were demonstrated tocontain FUM1 and FUM8 genes and also produce fumonisins in CMK medium.Fumonisins production tests indicated that the ability of fumonisin production variedsignificantly among different species or different strains within the same Fusariumspecie.The fumonisin level of fumonisin-producing isolates was from 186μg/g to16784μg/g （average,4637μg/g）;FB₁ is the primary component and up to 4041μg/g inaverage.In addition,the occurrence of fumonisin-producing isolates was differentamong corn samples from the three different areas,the most serious fungi andfumonisin-prouducer contamination was found in samples from eastern area.Ourresearch also suggested that fumonisin contamination was detected even in clean andhealthy edible corns,the colonization of fumonisin-producer likely occurred inpre-harvest,but fumonisin-production did not occure due to the unsuitable conditionsduring post-harvest.When the environment changed,these high-fumonisin-producingstrains might produce fumonisin.toxins in infected corn samples.The results obtained from HPLC-ELSD and LC-MS analyses showed that theasparagus samples from different areas of Zhejiang Province did not contain adetectable level of fumonisins.All asparagus samples collected for this study wereapparently clean and healthy.However,approximately 70% of the samples werecontaminated with Fusarium,including F.proliferatum,F.oxysporum,F.acuminatumand F.equesti.Among the fungal isolates,F.proliferatum accounted for over 50%.ThePCR results showed that fungal isolates contained the FUM genes,and also producedfumonisins in cultures,ranging from 28-4204μg/g culture media.Although multipleFusarium species were identified,our results showed that F.proliferatum was the onlyfumonisin-producing contaminant in the asparagus samples,although other Fusariumspecies might produce other different class of mycotoxins.Despite the fact that theasparagus from Zhejiang Province do not have apparent fumonisin contamination,there is a potential risk for human consumption of the asparagus spears due to the high frequency of natural occurrence of Fusarium spp,which could produce toxicmycotoxins,such as fumonisins produced by F.proliferatum.Among 15000 ethyl methanesulfonic acid-mutagenized Arabidopsis Col-0 seedssubjected to selection on 1μM FB₁,two FB₁-resistant （for） mutantsfbr41 and for42,which exhibited the most penetrant phenotypes,were identified and characterized infurther detail.In addition to resistance to FB₁,the mutants also showed rich root hair,hard leaf and less reactive oxygen species （ROS） production when grew on the FB₁medium and the tolerance of both mutants are much stronger than those of jar1-1,ein3-1,nprl mutants and transgenic NahG plants.Moreover,they display altered plantarchitecture in the absence of FB₁,such as dwarf,enhanced leaf margin serration anddeveloped lateral roots.A chi-square goodness-of-fit test confirmed that the fbr41phenotype of the F2 progeny segregated at the expected 3:1 （sensitive:resistant） ratiofor a single recessive mutation.The glucosinolates content of leaves in the Arabidopsis wild-type Col-0 wereanalyzed by HPLC after leaf infiltration with 10μM FB₁.The results showed that thecontent of short-chain aliphatic glucosinolates decreased,while the content oflong-chain aliphatic glucosinolates increased.Similarly,the content of indol-3-ylmethylglucosinolate （IM） decreased,while the content of 4-methoxyindol-3-ylmethylglucosinolate （4IM） and 1-methoxyindol-3-ylmethylglucosinolate （1IM）increased significantly.The observations that 4IM and IM were also induced in bothglucosinolate mutants gcc8 and gsml-3,and gsml-3 displayed a better resistance toFB₁ than Col-0,indicated that 4IM and 1IM instead of long-chain aliphaticglucosinolates might be involved in the resistance of Arabidopsis to FB₁.Geneexpression analysis suggested that no significant difference was observed between FB₁treatment and control in expression of NIT2 and PAD3,which were involved in IAAand camalexin biosynthesis,respectively.On the contrary,CYP83B1 was greatlyup-regulated by FB₁ treatment.The content of 4IM and 1IM is higher in both fbrmutants than that of wild-type Col-0.These results indicated that the biosynthesis of4IM and IM might be related to the resistance to FB₁ in Arabidopsis.The mechanisminvolved remains to be further elucidated.更多还原