表观遗传试剂应用于截短侧耳素产生菌Pleurotus mutilus-04的初步研究

发布时间：2018-01-01 03:37

本文关键词：表观遗传试剂应用于截短侧耳素产生菌Pleurotus mutilus-04的初步研究　出处：《西南大学》2011年硕士论文　论文类型：学位论文

【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。
[Abstract]:Background: with the excessive use of antibiotics in recent years, produced a lot of resistant bacteria to humans for many common diseases, so to find new compounds at a loss what to do, to promote the development of small molecule drugs imminent. Methods using the modified enzyme inhibitor of fungal chromosome provides a fast and effective mining fungal natural products in its own cell method, this method don't need expensive equipment, can be widely used in many laboratories, low cost, does not need a large workload and genetic background of fungi with no high requirements. In recent years epigenetic research, fungal epigenetic modification research has become a hot topic. Epigenetics refers to the chromosome in the DNA sequence does not change under the condition of gene expression has undergone genetic changes, including DNA methylation and its molecular mechanism, egg group The white modification, chromatin remodeling and RNA interference in several aspects such as research in fungi is much DNA methylation, histone methylation and acetylation. At present, the concept of genetic research means of fungal overexpression or deletion is mainly related to epigenetic modification of genes or by small molecule epigenetic reagent two aspects the secondary metabolites of fungi. Epigenetic modifications may alter the fungal spectrum, not only can improve many known secondary metabolites production, but also can activate silent biosynthetic gene clusters produced new metabolic products.
Objective: this experiment mainly through two kinds of small molecular epigenetic reagent vorinostat (suberoylanilide hydroxamic, acid, SAHA) - histone acetylation reagent and 5- azacytidine - DNA methyltransferase inhibitor to a strain capable of producing pleuromutilins large burden Pleurotus mutilus-04 phylum of fungi strain improvement was observed. Small molecular epigenetic reagents P utilus-04, the colony and mycelial morphology changes, changes in the secondary metabolic product spectrum.
Methods: this experiment by 96 hole plate method for the determination of two epigenetic reagent MIC value.P.mutilus-04 a day after inoculation with 2 times MIC epigenetic reagent, fermentation for 7 days, using a microscope to observe the mycelium by HPLC on the secondary metabolites, and use the preparation high performance liquid chromatography for separation of compounds, finally using NMR and mass spectrometry to analyze the structure of compounds.
Results: the study found that vorinostat P.mutilus-04, discovered the compound A intracellular by UV scanning and pop spectrum analysis, to determine the new compound A; isolated vorinostat analogues - compound D in fermentation broth, the activity detection of the anticancer activity of D compounds was significantly lower than that of vorinostat. Vorinostat experimental pleuromutilin yield relative to the yield of pleuromutilin negative control group decreased six times group of bacteria, while 5- azacytidine in experimental group pleuromutilin yield relative to the yield of pleuromutilin negative control group did not change. Although 5- complex secondary metabolites of 5-azacytidine on P.mutilus-04 had no significant effect, but has obvious effect on P.mutilus-04 colony morphology by 5- azacytidine treated P.mutilus-04, bacteria colony radius is large, thin wire Sparse.
Conclusion: vorinostat and 5- azacytidine two apparent colony genetic reagents on the large burden P.mutilus-04 phylum of fungi mycelial morphology, have different effects on the secondary metabolites, proved that this method can not only convenient in Penicillium Aspergillus, can also strain improvement in large burden in the phylum of fungi and verify the application of this method widely.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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