【作者】 沈硕；

【导师】 谢联辉； 欧阳明安；

【作者基本信息】 福建农林大学 ， 植物病理， 2009， 博士

【摘要】 从福建沿海海滩采集的各种海洋生物样品中分离纯化得到218株海洋真菌,其中酵母菌有52株。经液体发酵后,分别通过半叶法和叶碟法进行海洋真菌发酵液抑制烟草花叶病毒(Tobacco mosaic virus, TMV)侵染、抑制TMV增殖的活性筛选,通过MTT法进行发酵液抗肿瘤的活性筛选。结果表明,有39株海洋真菌的发酵液对TMV侵染的抑制率超过90%,6株海洋真菌的发酵液对TMV增殖的抑制作用较高,5株海洋真菌对两种肿瘤细胞的抑制活性均较好。进一步,对活性菌株进行了形态鉴定,并对其ITS rDNA序列进行了同源进化关系分析。结果表明,菌株1008F1 597kb的ITS rDNA序列与Neosartaya fischery(AF 176661)的ITS序列同源性高达100%,可鉴定为Neosartaya fischery.菌株1002F2575kb的ITS rDNA序列与Aspergillus sp.(EU159210、EU159210和EU159210)的ITS序列同源性高达100%,1009F1 609kb的ITS rDNA序列与Whalleya microplaca(EF026129)的ITS序列同源性高达98%,可鉴定为Whalleya microplaca.以7种植物病原真菌为供试菌种,对两株海洋真菌及其次级代谢产物的水溶性提取物进行抑菌试验。结果表明,菌株1002F2和菌株1009F1抑菌活性相对较高。其中,菌株1002F2水溶性提取物对高粱炭疽菌和番茄早疫菌的抑制活性较高,ECso值分别为1.34g/L和0.94g/L；菌株1009F1水溶性提取物对瓜果腐霉菌和番茄早疫菌的抑制活性较高,ECso值分别为0.63g/L和0.65g/L。对初筛得到的7株活性菌株的固体发酵产物粗提物进行抗TMV增殖活性的复筛,结果表明,菌株0312F1和1008F1的固体发酵产物活性仍然较高,从而将其确定为大量发酵的活性菌株。活性追踪的结果显示,菌株1008F1发酵产物甲醇提取物的活性部位在水溶性部分。采用柱层析(DCC)、薄层层析(TLC)和高效液相色谱(HPLC)等分离手段,从菌株1008F1(Neosartaya fischery)的水溶性提取物中共分离到9个纯品化合物。经波谱学鉴定结构,其中2个为蒽醌类化合物,化合物2个为腺苷类化合物,3个为生物碱类化合物。分别利用叶碟法和MTT法进行了抗TMV增殖和抗肿瘤活性的测定,对活性较高的化合物进行了抑制中浓度(EC50)的测定。结果显示,在200μg/mL供试浓度下,化合物3、5和8抗TMV增殖活性较高,EC50分别为153.08μg/mL、83.80μg/mL和96.99μg/mL；化合物5对胃癌细胞和肝癌细胞的抑制活性均较高,EC50分别为91.15μg／mL和100.02μg／mL。在此基础上,对化合物抗病毒及抗肿瘤活性构效关系(Structure-activity relationship, SAR)进行了分析。结果表明,抗植物病毒是生物碱类化合物的又一新的生物活性。就化合物5-7而言,C-7位羟基的存在是化合物5和7具有较高活性的原因,同时,C-7和C-8位之间双键的存在可能是化合物6和7活性降低的原因。更多还原

【Abstract】 There were 218 marine fungi isolated from marine organisms at beach in Fujian, China,52 of which were saccharomycetes. Inhibitory activities of fermentation broth of marine fungi against TMV infection and replication was determined by half-leaf and leaf-disc methods, respectively. And anti-tumor activity of fermentation broth of marine fungi was determined by MTT methods. There were 39 marine fungi, the inhibition rate of the fermention broth of which against TMV replication were over 90%. There were also 6 marine fungi, the inhibitory activity of the fermention broth of which against TMV replication was higher. And there were 5 marine fungi, the inhibitory activity of the fermention broth of which against both cancer cells proliferation was higher.Then, active strains were identified by morphology characterization. The homology relationships between active fungi and other fungi from GenBank were deduced from sequences of ITS rDNA. The results showed that 1008F1 was identified as Neosartorya fischeri, 1002F2 was identified as Aspergillus sp., and 1009F1 was identified as Whalleya microplaca.Seven plant pathogenic fungi were selected for test. The inhibitory activity of 3 marine fungi and their water-soluble fraction of the secondary metabolites against plant pathogenic fungi were evaluated. The results showed that the inhibitory activities of water-soluble fraction of strain 1002F2 against Colletotrichum graminicola and Alternaria solani were higher, with EC50 values 1.34 g/L and 0.94 g/L. And the inhibitory activities of water-soluble fraction of strain 1009F1 against Pythium aphanidermatum and Alternaria solani were higher, with EC50 values 0.63 g/L and 0.65 g/L,Further screening test about anti-TMV replication was carried on crude extracts of 7 strains which showed more active after preliminary screening test. Strain 0312F1 and 1008F1 were determined as active strains for further test because of their high activity. Guided by anti-tumor activity, the results showed that the active fraction named 1008F1 was water-soluble.Nine compounds were isolated from strain 1008F1 by DCC, TLC and HPLC instrumentations. According to the chemical structures identification,2 compounds were anthraquinones,2 compounds were adenosines and 3 compounds were alkaloids. Inhibitory activities of compounds against TMV replication and cancer cells proliferation were determined by leaf-disc and MTT methods, respectively. Then, the EC50 values were examined on compounds showed higher activity. In the tested concentration, the anti-TMV replication activity of compounds 3,5 and 8 was much higher, with EC50 values 153.08μg/mL,83.80μg/mL and 96.99μg/mL, respectively. Anti-tumor activity of compound 5 against both cancer cells was higher, with EC50 values 91.15μg/mL and 100.02μg/mL. In addition, the structure-activity relationship (SAR) between structures of active compounds and anti-TMV replication and anti-tumor activity was analyzed. It was shown that anti-phytovirus activity was another new bio-activity about alkaloids. Take compounds 5-7 for example, maybe hydroxyl at C-7 inhanced the activity of compounds 5 and 7. And double bond between C-7 and C-8 perhaps decreased the activity of compound 6.更多还原