【作者】 周莹；

【导师】 汪俏梅；

【作者基本信息】 浙江大学 ， 蔬菜学， 2007， 硕士

【摘要】 腐马素是一组主要由串珠镰刀菌（Fusarium verticillioides或Fusariummoniliforme）和再育镰刀菌（Fusarium proliferatum）产生的真菌毒素。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，严重威胁食品安全及人类和动物的健康。串珠镰刀菌和再育镰刀菌是世界范围内常见的作物和食品的污染菌，在玉米和芦笋上也有着广泛的侵染。本研究建立了一种经济、快速、简便、准确的腐马素产毒株的复合PCR鉴定方法和快速测定玉米产品中B族腐马素的HPLC-ELSD法，并分析了我国各地收集玉米样品上，浙江省内芦笋样品上腐马素产毒株的侵染状况，对样品上分离菌株的腐马素产毒力也进行了测定。研究结果如下：以串珠镰刀菌野生型的聚酮化合物合成酶（PKS）基因FUM1，丝氨酸-十六酰基转移酶基因FUM8，长寿保证因子基因FUM17核苷酸序列为基础，检索GenBank数据库，采用DNAman软件，分别设计了多对引物。以串珠镰刀菌野生型菌株（A0149）（FGSC号7600）为阳性对照，以PKS被敲除的串珠镰刀菌野生型菌株和桔青霉等菌株为阴性对照。以特异性高的引物对fum1-2L／fum1-2R，fum8-3N／fum-8-4E，fum17-P-F2／fum17-P-R2与镰刀菌属特异的引物对核糖体rDNA及其内转录间隔区（ITS）序列引物ItsR/ItsF（Bluhm et al.,2002）结合，建立了串珠镰刀菌腐马素产毒菌株复合PCR检测方法，检出限为1ng。使用分别基于FUM1，FUM8的引物rp32／rp33，rp679／rp680（Proctor et al.,2004）及ItsR／ItsF引物，建立了腐马素产毒菌株的复合PCR检测方法，检出限为100pg。在44份未加工玉米样品的21份上分离菌株25个，在16个芦笋样品上分离菌株16个，对此41个菌株用上述方法进行了分析，得到的25个PCR检测阳性菌株（玉米上15株，芦笋上10株）。经形态学鉴定及蛋白质翻译延伸因子（TEF）基因PCR产物测序鉴定后，确定玉米上分离的菌株均为串珠镰刀菌，芦笋上分离的菌株均为再育镰刀菌。同时，建立了快速测定玉米培养物中B系列腐马素的HPLC-ELSD法，无需衍生，快速、准确地测定B族腐马素含量和组成，检出限为6ng。具有较好的定量灵敏度。并应用该法对玉米及芦笋样品上分离菌株的腐马素产毒能力进行了测定。结果显示，所有样品上分离的产毒基因PCR检测阳性镰刀菌菌株在玉米培养基上均能产生FB₁（125-40321 μg/g），芦笋上分离的再育镰刀菌中，除了一个菌株为中等产毒菌株（50 μg/g至500 μg/g）外，其余全部为高产毒株（≥500μg/g）。不同菌株产毒能力差异也比较大，串珠镰刀菌不同菌株产生FB₁浓度范围约为1331至40321μg/g，再育镰刀菌产生FB₁浓度范围为125-19251μg／g。另外，大部分菌株还能产生FB₂及FB₄，部分菌株能产生FB₃，且所有菌株产生FB₁的量都高于FB₂，FB₃及FB-4的量。更多还原

【Abstract】 Fumonisins are a group of mycotoxins mainly produced by Fusahum vertici-llioides （formerly F. moniliforme） and Fusarium proliferatum. Fumonsins have been demonstrated to cause several fatal diseases in animals, such as leukoencephalomala-cia in horses, pulmonary oedema and hydrothorax in swine and hepatic cancer in rats. In humans, fumonisins are associated with oesophageal cancer and birth neural tube defects. F. verticillioides and F. proliferatum are found worldwide and have been reported in maize, asparagus, and many other agriculture products.We have developed an economical, rapid, simple, and precise multiplex PCR method for identifying fumonsin-producing fungal strains in corn and asparagus. We have also developed a method for direct quantification of B-series fumonsins （FBs） in corn products. This method is based on high-performance liquid chromatography coupled with an evaporative laser scattering detector （HPLC- ELSD）, which allows direct detecting the non-UV-absorbing fumonisins without any prior derivatization of the samples. The method ensures accurate quantification of FBs with a limit of 6 ng/μl.Using the methods, we have studied the occurrence of fumonsin-producing strains in maize grain samples from household and supermarket of limited areas in China and in edible asparagus from Zhejiang Province. Furthermore, the ability of the isolated srains to produce FBs on sterile maize grain has been determined. The results of the studies are as follows:Several pairs of primers that are specific for fumonsin-producing strains were designed. These primers, including fuml-2L/fuml-2R, fum8-3N/fum-8-4E, fum17-P-F2/fuml7-P-R2, rp32/rp33, and rp679/ rp680 （Proctor et al., 2004）, were based on the polykefide synthase （PKS） gene （FUMI）, serine-palmitoyltransferase gene （FUM8）, and longevity assurance factor （also known as ceramide synthase） gene （FUMI7） that are involved in fumonisin biosynthesis. In addition, Fusarium genus-specific primers, ItsR/ItsF （Bluhm et al., 2002）, were also prepared. With both fumonisin-specific primers and genus-specific primers, Multiplex PCR were carried out to detect fumonsin-producing strains and Fusarium verticllioides in the food products collected from various areas in China. As control experiments, the wild-type fumonisin-producing strain A0149 （FGSC NO.7600） of F. verticillioides, the PKS-deleted non-fumonisin-producing mutant, and Penicillium citrinum were included in the multiplex PCR. The detection limits of the multiplex PCR detecting fumonisin-producing Fusarium verticillioides with primers fuml-2R, fum8-3N/fum-8-4E, fum17-P-F2/fum17-P-R2 and ItsR/ItsF and the multiplex PCR detecting fumonisin-producing strains with primers rp32/rp33, rp679/rp680 and ItsR/ItsF were 1 ng and 100 pg template DNA per PCR assay respectively.Among 58 corn samples, 21 samples contained fungal strains （total 25 isolates）; from 16 edible asparagus samples, 16 isolates were identified. The 41 isolates were analyzed with the multiplex PCR, and the results showed that 25 were fumonisin-producing Fusarium strains （15 from corn and 10 from asparagus）. The positive strains were subjected to further morphological identification, as well as molecular identification by analyzing the sequence of PCR amplified translation elongation factor-1α（TEF）. The results showed that all the isolates from corn are F. verticillioides and all the isolates from asparagus were F proliferatum.The ability of the positive isolates identified by PCR to produce fumonisins was examined by HPLC-ELSD. All 25 isolates produced FB₁ （125-40321μg/g） on autoclaved maize kernels. The 10 asparagus isolates were all high producer （≥500μg/g） except one isolate with a modest yield （50μg/g-500μg/g）. The ability to produce FBs differed among different isolates of the same species. For the F. verticillioides isolates, the yield of FB₁ varied from 1331 to 40321μg/g, while for F. proliferatum isolates the yield of FB₁ from 125 to 19251μg/g. Most isolates also produced FB₂ and FB₄, while some also produced FB₃. In all isolates, the yield of FB₁ was always the highest among all fumonisins.更多还原