鸦胆子内生真菌Br11代谢产物抗TMV活性研究

发布时间：2018-10-09 13:34

【摘要】：世界上最早被发现的病毒就是烟草花叶病毒(Tobacco msaic virus,TMV),TMV严重影响着我国烟叶的产量和质量,每年对我国烟草的生产造成巨大的损失,但是到目前为止并没有一种可以根治烟草花叶病毒(TMV)的药剂。前期已有研究者从药用植物鸦胆子中分离出一种对烟草花叶病毒(TMV)有很好的抑制作用的化学物质—鸦胆子素D(BruceineD),基于植物内生真菌与宿主之间互利共生的关系,植物内生真菌能够产生与宿主相同或相似的化合物,研究室早期对鸦胆子植物中的内生真菌进行了分离纯化,并对这些真菌的代谢产物进行抗TMV活性的粗测,本文选取其中一株较好的抗TMV的内生真菌Br11进行深入的研究。本文对鸦胆子植物的一株内生真菌Br11进行代谢产物分离及其抗TMV活性的研究。发酵该菌株,利用多种色谱分离技术如硅胶柱层析、RP-C18层析柱、SephadexLH-20葡聚糖凝胶柱层析等对其发酵产物进行分离纯化,得到6种单体化合物和一个胞外多糖(Extracellular polysaccharide,EPS),分别编号为 1-7。通过1H-NMR、13C-NMR波普技术分析,对这6种化合物结构进行鉴定及查阅文献对比,得出化合物1-6分别为DothioreLone A、Dothiorelone B、Dothiorelone C、Dothiorelone H、Cytosporone C、Cytosporone U。对这些分离出来的化合物进行了抗TMV活性研究,采用半叶枯斑法和叶碟法分别测定这几种化合物对TMV侵染和增殖的抑制活性。结果表明胞外多糖(组分7)有很强的抗TMV侵染效果,其IC50为0.93μg/mL,但抗TMV增殖效果不明显。化合物6 Cytosporone U有较强的抗TMV增殖的作用,其IC50为144.60μg/mL。采用Western-blot和农杆菌浸润法进一步测定化合物6在烟草植株内抗TMV的生物活性。结果发现在浓度为200 μg/mL时,化合物6起到很好的抗TMV-CP表达、抗TMV增殖和运动的效果,其有效时期为7天。本文通过形态学与分子生物学相结合的方法对Br11菌株进行了鉴定,该真菌在PDA培养基上菌落生长迅速,菌丝丝状长管,有隔,颜色由白色变为淡黄色再变为深黄色,并产生了黄色的可溶性色素。采用内生真菌通用引物ITS 1和ITS 4对Br11菌株的28S rRNA的保守区序列进行PCR扩增并对扩增产物进行序列测定,通过BLAST分析序列的同源性,发现该菌株与拟茎点霉属同源性最高,为99%。通过构建系统发育树,发现Br11菌株与拟茎点霉属在同一分支上。同时对Br11菌株进行了不同培养条件下的诱导产孢实验,均没有发现Br11菌孢子的形态。综合以上及相关文献描述,确定Br11菌株为不产孢的拟茎点霉(Phomopsis sp)真菌。
[Abstract]:The earliest virus found in the world is tobacco mosaic virus (Tobacco msaic virus,TMV), which seriously affects the yield and quality of tobacco in China, and causes huge losses to the production of tobacco in China every year. But so far there is no cure for tobacco mosaic virus (TMV). Brucetin D (BruceineD), a chemical substance that has been isolated from Brucea javanica, a medicinal plant, which has a good inhibitory effect on tobacco mosaic virus (TMV) (TMV), is based on the mutually beneficial symbiotic relationship between endophytic fungi and the host. Plant endophytic fungi can produce the same or similar compounds as the host. The endophytic fungi from Brucea javanica plants were isolated and purified in the early stage, and the metabolites of these fungi were tested for their anti- TMV activity. In this paper, one of the better endophytic fungi Br11 resistant to TMV was selected for further study. In this paper, the metabolites of an endophytic fungus Br11 from Brucea javanica were isolated and its anti TMV activity was studied. The fermentation products of the strain were separated and purified by a variety of chromatographic separation techniques, such as RP-C18 column Sephadex LH-20 gel column chromatography. Six monomers and one extracellular polysaccharide (Extracellular polysaccharide,EPS) were obtained, numbered 1-7, respectively. By 1H-NMR-13C-NMR technique, the structures of the six compounds were identified and compared in literature. The results showed that compounds 1-6 were DothioreLone Dothiorelone (C) Dothiorelone (H) and Cytosporone (C) to Cyporone (U). The anti- TMV activity of these isolated compounds was studied. The inhibitory activities of these compounds on TMV infection and proliferation were determined by semi-leaf blight spot method and leaf disc method respectively. The results showed that the extracellular polysaccharides (component 7) had a strong anti-infection effect of TMV, its IC50 was 0.93 渭 g / mL, but the anti-proliferation effect of TMV was not obvious. Compound 6 Cytosporone U had a strong anti-proliferation effect on TMV, with a IC50 of 144.60 渭 g / mL. The bioactivity of compound 6 against TMV in tobacco plants was further determined by Western-blot and Agrobacterium tumefaciens infiltration method. The results showed that compound 6 had a good effect on the expression of TMV-CP, proliferation and movement of TMV at a concentration of 200 渭 g/mL. The effective period of compound 6 was 7 days. In this paper, the Br11 strain was identified by combining morphology and molecular biology. The fungus grew rapidly on the PDA medium. The mycelium had long tube and septum, and the color changed from white to light yellow and then to dark yellow. Yellow soluble pigment was produced. Endophytic fungi universal primer ITS 1 and ITS 4 were used to amplify the conservative region sequence of 28s rRNA of Br11 strain by PCR and sequencing the amplified products. The homology of BLAST analysis showed that this strain had the highest homology with P. pseudostema, which was 99xuan. By constructing phylogenetic tree, it was found that the Br11 strain was on the same branch as the phyllostachys. At the same time, the spore morphology of Br11 strain was not found in the spore induction experiment under different culture conditions. According to the above and related literatures, Br11 strains were identified as (Phomopsis sp) fungi without sporulation.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476

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