【作者】 周锐；

【导师】 胡昌华；

【作者基本信息】 西南大学 ， 微生物与生化药学， 2011， 硕士

【摘要】 背景：随着近些年来抗生素的过度使用,产生了很多耐药菌,使人类对众多常见疾病束手无策,因此寻找新型化合物促进药物开发迫在眉睫。采用小分子酶抑制剂修饰真菌染色体的方法提供了一个快速有效的在其自身细胞内挖掘真菌天然产物的方法,该方法不需要昂贵的仪器设备,可以广泛的在许多实验室中应用,成本较低,不需要较大工作量且对真菌的遗传背景无过高要求。随着近些年来表观遗传学研究的深入,真菌的表观遗传修饰研究逐渐成为热点。表观遗传学就是指在染色体中DNA序列不发生变化的情况下,基因表达却发生了可遗传的改变,其分子机制主要包括DNA甲基化、组蛋白修饰、染色质重塑以及RNA干扰等几个方面,在真菌中研究比较多的就是DNA甲基化、组蛋白甲基化和乙酰化。目前,真菌的表观遗传研究手段主要是过表达或缺失相关表观修饰基因或利用小分子表观遗传试剂两个方面。真菌进行表观遗传修饰可能会改变真菌的次级代谢产物谱,不仅可以提高多种已知次级代谢产物的产量,而且可以激活沉默生物合成基因簇产生新的代谢产物。目的：本实验主要通过两种小分子表观遗传试剂伏立诺他(suberoylanilide hydroxamic acid, SAHA)—组蛋白去乙酰化试剂和5-杂氮胞苷—DNA甲基转移酶抑制剂来对一株能够产生截短侧耳素的大型担子纲真菌Pleurotus mutilus-04进行菌种改良。观察小分子表观遗传试剂作用P utilus-04后,其菌落、菌丝形态的变化以及其次级代谢产物谱的变化。方法：本实验通过96孔板方法测定两种表观遗传试剂的MIC值。P.mutilus-04接种一天后加入2倍MIC表观遗传试剂,发酵培养7天后,使用显微镜对其菌丝进行观察,采用高效液相色谱对其次级代谢产物进行分析,并使用制备型高效液相色谱对化合物进行分离,最后利用核磁共振仪及质谱仪对化合物进行结构解析。结果：研究发现伏立诺他作用P.mutilus-04后,胞内发现了化合物A,进行了紫外波普扫描以及质谱分析,确定其为新化合物A；在其发酵液中分离得到了伏立诺他的结构类似物-化合物D,活性检测发现化合物D的抗癌活性明显低于伏立诺他。伏立诺他实验组菌体中截短侧耳素的产量相对于阴性对照组中截短侧耳素的产量下降了六倍,而5-杂氮胞苷实验组中截短侧耳素的产量相对于阴性对照组中截短侧耳素的产量没有较大变化。虽然5-杂氮胞苷对P.mutilus-04的次级代谢产物没有明显的影响,但其对P.mutilus-04的菌落形态有较明显的影响,经5-杂氮胞苷处理后的P.mutilus-04,菌落半径较大,菌丝稀疏。结论：伏立诺他和5-杂氮胞苷两种表观遗传试剂对大型担子纲真菌P.mutilus-04的菌落、菌丝形态,次级代谢产物等方面有不同程度的影响,证明这种快捷简便的方法不仅可以在青霉曲霉中,也可以在大型担子纲真菌中进行菌种改良,验证了这种方法的应用广泛性。更多还原

【Abstract】 Background:As in recent years, epigenetic research in tumor cells growing in depth, many microbiologists gradually introduced to the discipline on the fungal secondary metabolite research. Epigenetics refers to DNA sequences in the chromosomes does not change, gene expression is heritable changes have taken place, and its molecular mechanisms including DNA methylation, histone modification, chromatin remodeling and RNA interference. In fungi, more research is about DNA methylation, histone methylation and acetylation. At present, By over-expressing or deleting the epigenetic modified genes and using small molecule epigenetic modifying agents to change fungal chromosome modified forms, we can improve production of microbic secondary metabolites and activate the silent biosynthesis gene clusters successfully. With the excessive use of antibiotics in recent years, resulting in a lot of drug-resistant, many common diseases can not be cured by existing drugs, so search for new compounds for drug development is imminent. using small molecules to manipulate the fungal epigenome provides a needed tool for rapidly accessing potential pools of cryptic fungal natural products in their native hosts.Second, this methodology can be readily implemented in most labs without extensive retooling, giving it a wide scope of utilization.Third, this approach will significantly lessen the cost and effort of acquiring the products of silent secondary metabolic pathways since fungi do not need to be pre-screened using a multitude of culture conditions.Aim:In this study, by two small-molecule epigenetic agents suberoylanilide hydroxamic acid and 5-azacytidine on Pleurotus mutilus-04 for strain improvement. Observed the changes of P.mutilus-04 colonies, mycelium morphology and its secondary metabolites by useing small-molecule epigenetic agents.Methods:This experiment determines the MIC of two kind of small-molecule epigenetic agents by using 96 well plate. P.mutilus-04 cultured for one day,added-on 2 time MIC of the epigenetic agents, and then fermentation for 7 days.We use the microscope to carry on the observation to its mycelial, use the highly effective liquid chromatography to carry on the analysis to its secondary metabolites, and use the preparation highly effective liquid chromatography to separate compound A and D. Finally it carries on the structure analysis using the nuclear magnetic resonance spectrometer and the mass analyzer to the compound.Result:P.mutilus-04 modified with SAHA,its mycelial、colony morphology had no change, the compound A never produced in this fungi was discovered in intra-cellular, the compound A was scanned by UV and mass spectrometry analysis. In its fermentation broth, compound D had been Purification, which is a structural analogue of SAHA. The anticancer activity of compound D was significantly lower than SAHA. The production of pleuromutilin is six times lower than negative control in SAHA experimental group. However,there is no major changes between 5-azacytidine experimental group and negative control. Although it had no signigicant effect in secongdary metabolites of P.mutilus-04 by 5-azacytidine,but 5-azacytidine on colony morphology of P.mutilus-04 have a more significant impact, the colony radius was larger and myceliums were sparse.Conclusion:The colony morphology, mycelium morphology and secondary metabolites of P.mutilus-04 have different effects by SAHA and 5-azacytidine.It proves that the method can making Strain improvement not only in Aspergillus and Penicillium, but also in basidiomycetes. So this method has broad application.更多还原