IOCAS-IR
海洋细菌源生物农药前体的筛选及抗菌机制研究
张林林
学位类型博士
2018-05-04
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
学位名称理学博士
关键词农业病原菌,海洋微生物,吩嗪-1-羧酸,丰原素,抗菌机理
摘要

由农业病原菌引起的一系列农业灾害严重威胁我国粮食生产安全,导致巨大的经济损失。微生物源生物农药因为具有低毒性,环境友好性等优点而受到越来越多的关注。作为世界上最大的生态系统,海洋环境中蕴含着多样的微生物和活性物质,为新型生物农药前体的开发提供了丰富的候选资源。因此,本研究拟开展两个方面的研究,一方面以病原细菌鳗弧菌和病原真菌烟草黑胫病菌为研究对象;另一方面,以病原真菌稻瘟病菌为研究对象,筛选拮抗这些病原菌的海洋细菌及其活性产物,并利用蛋白组学和分子生物学等技术研究其抗菌机理。

吩嗪及其衍生物是由假单胞菌所产生的非常重要的次级代谢产物,并且具有广谱的拮抗细菌和真菌的活性。但是直到现在,有关来自海洋环境的假单胞菌及其吩嗪类产物却鲜有报道。在本研究中,一株具有拮抗病原细菌的海洋源细菌Pseudomonas aeruginosa PA31x被分离出来,该菌株可以抑制水产养殖业中危害最严重的病原细菌之一鳗弧菌(Vibrio anguillarum)的生长。利用高分辨率质谱以及核磁共振图谱分析了该菌株所产的功能性产物,将该活性产物鉴定为吩嗪类重要的衍生物,吩嗪-1-羧酸(PCA)。结合基因克隆等分子生物学手段发现P. aeruginosa PA31x菌株的基因组中含有两个合成PCA的基因簇,分别为phz1phz2。电子显微镜观察后发现经过PCA处理过的鳗弧菌C312细胞壁出现了破裂,并且伴随着细胞质泄露到周围环境。其他的实验结果显示PCA也可以诱导病原菌以及癌细胞胞内活性氧(ROS)的积累,继而导致细胞死亡。值得注意的是,本实验中发现PCA可以显著降低鳗弧菌C312对斑马鱼卵的感染。而且,在温室防控实验中,PCA可以明显降低烟草黑胫病菌对烟苗的感染。综合上述实验结果,本研究揭示了海洋铜绿假单胞菌来源的PCA可以显著抑制水产养殖业以及农业病原菌,扩展了PCA在农业防治领域中的应用范围。

由病原真菌稻瘟病菌引起的稻瘟病是一种严重危害世界粮食安全和非常难以防控的农业病害。芽孢杆菌已经被广泛的应用于很多病原真菌的生物防控。在本研究中,海洋细菌Bacillus subtilis BS155对稻瘟病菌的生长显示出显著地抑制活性。通过高效液相(HPLC),电喷雾离子化质谱法(ESI-MS)和串联质谱法(ESI-MS/MS),对该菌株的活性产物进行分离,并最终鉴定为丰原素类脂肽,命名为fengycin BS155。通过使用扫描和透射电子显微镜进一步在亚细胞水平揭示了fengycin BS155所引起的稻瘟病菌菌丝细胞的细胞壁以及质膜的形态学变化。利用蛋白组学以及生物化学手段,发现fengycin BS155可以显著地降低线粒体的膜电位(MMP),诱导胞内活性氧的大量积累(ROS)以及降低ROS清除相关酶的活性。同时,fengycin BS155也会引起稻瘟病菌菌丝胞内染色体的皱缩,导致DNA修复相关酶的蛋白表达水平升高和多聚(ADP核糖)聚合酶[poly ADP-ribose polymerasePARP]的剪切。综上所述,本实验结果表明fengycin BS155可以诱导稻瘟病菌膜损伤和细胞器的功能紊乱,降低线粒体膜电位,产生氧化压力和引起染色体皱缩,最终导致稻瘟病菌菌丝细胞的死亡。因此,fengycin BS155及其产生菌株在防控稻瘟病菌及其引发的稻瘟病中有非常好的应用潜力,且需要进一步开展其应用工作。

 

其他摘要

A series of agricultural dieases and disasters caused by agricultural pathogens seriously threaten food production safety and cause great economic loss in China. Microbial source biological pesticides have attracted more and more attention due to its low toxicity and environmental friendliness. As the largest ecosystems in the word, marine environment has a variety of microbial strains and active material resources. Marine environment is rich in the resources of active compound candidates and provides novel biological pesticide precursors. In our research, the agricultural pathogenic microorganisms, including Vibrio anguillarum, Phytophthora nicotianae and Magnaporthe grisea, were used as the research object to obtain the antagonistic bacteria and their active products. Furthermore, the antimicrobial mechanisms of obtained active compounds were also explored with proteomic and molecular biology techniques.

Phenazine and its derivatives are very important secondary metabolites produced from Pseudomonas spp. and have exhibited broad-spectrum antifungal and antibacterial activities. However, till date, there are few reports about marine derived Pseudomonas and its production of phenazine metabolites. In this study, we isolated a marine strain Pseudomonas aeruginosa PA31x which produced natural product inhibiting the growth of V. anguillarum, one of the most serious bacterial pathogens in marine aquaculture. Combining high-resolution electro-spray-ionization mass spectroscopy and nuclear magnetic resonance spectroscopy analyses, the functional compound against V. anguillarum C312 was demonstrated to be phenazine-1-carboxylic acid (PCA), an important phenazine derivative. Molecular studies indicated that the production of PCA by P. aeruginosa PA31x was determined by gene clusters phz1 and phz2 in its genome. Electron microscopic results showed that treatment of V. anguillarum C312 with PCA led complete lysis of bacterial cells with fragmented cytoplasm being released to the surrounding environment. Additional evidence indicated that reactive oxygen species generation preceded PCA-induced microbe and cancer cell death. Notably, treatment with PCA gave highly significant protective activities against the development of V. anguillarum C312 on zebrafish. Additionally, the marine derived PCA was further found to effectively inhibit the growth of agricultural pathogens P. nicotianae. Taken together, this study reveals that marine Pseudomonas derived PCA carries antagonistic activities against both aquacultural and agricultural pathogens, which broadens the application fields of PCA.

Rice blast caused by phytopathogen M. grisea posesses a serious threat to the global food security and is difficult to control. Bacillus species have been extensively explored for the biological control of many fungal diseases. In the present study, marine bacterium Bacillus subtilis BS155 showed a strong antifungal activity against M. grisea. The active metabolites were isolated and identified as cyclic lipopeptides (CLPs) of the fengycin family, named fengycin BS155, by the combination of high-performance liquid chromatography (HPLC), electrospray ionisation mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Analyses using scanning and transmission electron microscopy revealed that fengycin BS155 caused morphological changes in the plasma membrane and cell wall of M. grisea hyphae. Using comparative proteomic and biochemical assays, fengycin BS155 was demonstrated to reduce the mitochondrial membrane potential (MMP), induce bursts of reactive oxygen species (ROS) and down-regulate the expression level of ROS scavenging enzymes. Simultaneously, fengycin BS155 caused chromatin condensation in fungal hyphal cells, which led to the up-regulation of DNA repair-related proteins expression and the cleavage of poly (ADP-ribose) polymerase (PARP). Taken together, our results indicate that fengycin BS155 acts by inducing membrane damage and dysfunction of organelles, disruption of MMP, oxidative stress, and chromatin condensation, resulting in M. grisea hyphal cell death. Therefore, fengycin BS155 and its parent bacterium are very promising candidates for the biological control of M. grisea and the associated rice blast.

语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154433
专题中国科学院海洋研究所
第一作者单位中国科学院海洋研究所
推荐引用方式
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张林林. 海洋细菌源生物农药前体的筛选及抗菌机制研究[D]. 中国科学院海洋研究所. 中国科学院大学,2018.
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