【作者】 王红；

【导师】 夏志洁； 夏雪奎；

【作者基本信息】 山东师范大学 ， 微生物学， 2021， 硕士

【摘要】 菌核青霉可以产生出结构多种多样的次级代谢产物。这些次级代谢产物功能特殊,在抗菌、抗氧化、抗肿瘤等方面有着重要的作用。本论文利用第三代测序技术对菌核青霉SD-36(Penicillium sclerotiorum SD-36)进行了全基因组测序,通过生物信息学的方法对其基因组进行挖掘,预测其包含53条基因簇。通过表观遗传学手段对HDAC(组蛋白去乙酰化酶)进行敲除,因此激活了沉默基因簇,产生了两个吲哚类化合物。同时通过过表达特定双PKS基因簇的转录调节因子,使得该簇核心基因的表达量增加。本论文的研究和结果主要包括以下几个方面:1.基因组测序和生物信息挖掘本实验通过第三代测序技术对菌核青霉SD-36进行了全基因组测序,计算出其基因组总长度为37242374 bp,平均G+C含量为46.74%。通过anti SMASH程序在菌核青霉SD-36基因组中注释了53个次级代谢产物基因簇。这些基因簇包括PKS(聚酮合酶)、NRPS(非核糖体肽合成酶)、TCs(萜烯环化酶),indole(吲哚)、PKS-NRPS、TC-PKS、TC-NRPS和其它种类的酶。2.基因敲除突变株的产生和生长表型本实验使用hygromycin B(潮霉素B)抗性筛选出三个基因敲除突变体(P.sclerotiorum SD-36Δ1628-5,-6,-7),在相同的培养时间内,野生型菌株和突变菌株P.sclerotiorum SD-36Δ1628-5,-6,-7在PDA(马铃薯葡萄糖琼脂)培养基上的菌落在颜色以及孢子形态上有很大不同。野生型菌株呈现绿色,而突变株的颜色偏青。同时在PDA平板的背面显示了野生型菌株呈现黄色,比突变株的黄色更为显著。3.基因敲除突变株新化合物的产生及结构鉴定对野生型菌株和P.sclerotiorum SD-36Δ1628-5,-6,-7突变株的提取物进行了发酵实验和HPLC(高效液相)分析。与野生型菌株进行相比,三株突变株的次级代谢产物HPLC谱图发生了显著变化。从突变株提取物的HPLC谱图中观察到多个新峰(含峰1和峰2),我们最终分离出其中两种化合物并分别鉴定为meleagrin和roquefortine C(罗格福汀C)。4.过表达突变株的产生和生长表型本实验使用hygromycin B筛选了五株转录调节因子过表达突变株,在相同的培养时间内,野生型菌株和突变菌株P.sclerotiorum SD-36 OE::PsAza1-1,-2,-3,-4,-5在PDA培养基上的菌落在形态上以及颜色上有很大不同,野生型菌株呈绿色,而突变株是橙红色的。5.过表达突变株化合物的检测及结构鉴定对野生型菌株和P.sclerotiorum SD-36 OE::PsAza1-1,-2,-3突变株的提取物进行了发酵实验和HPLC谱图分析。与野生型菌株相比,三株突变株的次级代谢谱图发生了明显变化。从突变株提取物的HPLC谱图中观察到较野生株液相谱图产量增加的峰,我们最终分离出其中两种化合物并分别鉴定为嗜氮酮(azaphilones)类化合物isochromophiloneⅥ和sclerotiorin C。6.双PKS基因簇核心基因的转录水平上调qRT-PCR分析了WT株和OE::PsAza1突变株中3个核心基因(HR-PKS、NR-PKS、Psaza1)的表达水平。与野生株相比,HR-PKS,NR-PKS和转录因子Psaza1的表达上调60-80倍。本实验的创新点在于:1.通过基因敲除产生了HDAC缺失突变体,通过对突变株发酵液的HPLC分析出现了较野生株的新峰,通过对次级代谢产物的分离纯化并最终鉴定为两种吲哚生物碱:meleagrin和roquefortine C。据我们所知,这是通过在菌核青霉中调节HDAC蛋白来获得meleagrin和roquefortine C的第一个例子。我们的实验结果表明表观遗传修饰在指导真菌中生物活性天然产物的发现方面有着巨大的潜力。2.我们对菌核青霉SD-36进行基因挖掘找到特殊的双PKS基因簇,通过过表达转录调节因子PsAza1成功得到突变株,通过对突变株发酵液的提取物进行HPLC分离制备得到了两个较野生株产量增加的嗜氮酮类化合物,嗜氮酮类化合物具有广泛的生物学活性和令人感兴趣的结构特征,同时它具有良好的抗细胞毒性、抗病毒、抗微生物、抗炎以及抗癌活性,该类化合物越来越受到大量研究人员的重视。更多还原

【Abstract】 Penicillium sclerotiorum SD-36 can produce a variety of secondary metabolites.These secondary metabolites have special functions and diverse structures,which play important roles in antibacterial,anti-oxidant,and anti-tumor aspects.In this thesis,the third-generation sequencing technology was used to sequence the whole genome of P.sclerotiorum SD-36,and 53 gene clusters in the fungus were predicted by bioinformatics methods.Through epigenetic means,the silent gene clusters were activated by knocking out HDAC gene,and thus two indole compounds were produced.At the same time,the overexpression of a transcription regulator of a specific double PKS gene cluster up-regulated the expression of core genes on the cluster.The research results of this paper mainly include the following aspects:1.Genome sequencing and bioinformation miningIn this experiment,the whole genome of P.sclerotiorum SD-36 was sequenced through the third-generation sequencing technology.The total size of the genome was calculated to be 37242374 bp,and the average G+C content was 46.74%.53 gene clusters of secondary metabolites were annotated in the genome of P.sclerotiorum SD-36 using the anti SMASH program.These gene clusters include PKS,NRPS,TCs,indole,PKS-NRPS,TC-PKS,TC-NRPS and other types of gene clusters.2.Generation and growth phenotype of gene knockout mutantsIn this experiment,three gene knockout mutants(P.sclerotiorum SD-36 Δ1628-5,-6,-7)were generated using the hygromycin B selection marker system,In the same culture time,the colonies of wild-type strain and mutant strain P.sclerotiorum SD-36Δ1628-5,-6,-7 on PDA medium are very different in color and spore morphology.The wild-type strain is green,while the mutant strain is greenish.At the same time,the back of the PDA plate shows that the wild-type strain is yellow,which is a little more obvious than the yellow of the mutant strain.3.Generation and structure identification of new compounds.Fermentation experiments and HPLC analysis were performed on extracts of wild-type strains and P.sclerotiorum SD-36 Δ1628-5,-6,-7 mutant strains.Compared with the wild-type strain,the secondary metabolism profiles of the three mutant strains have undergone significant changes.Several new peaks(containing peak 1 and peak 2)were observed from the HPLC spectrum of the mutant extract.We finally separated two of the compounds and identified them as meleagrin and roquefortine C,respectively.4.Production and growth phenotype of overexpression mutant strainsIn this experiment,the hygromycin B selection marker system was used to generate three transcriptional regulator overexpression mutant strains.In the same culture time,The wild-type strain and the mutant strain P.sclerotiorum SD-36OE::PsAza1-1,-2,-3,4,5 have very different colonies on the PDA medium in morphology and color.The wild-type strain is green,while the mutant is orange-red.5.Increased production of compounds and structural identificationFermentation experiments and HPLC analysis were performed on extracts of wild-type strains and P.sclerotiorum SD-36 OE::PsAza1-1,-2,-3 mutant strains.Compared with the wild-type strain,the secondary metabolism profiles of the three mutant strains have undergone significant changes.From the HPLC spectrum of the mutant extract,an increase in yield was observed compared to the liquid phase spectrum of the wild strain.We finally separated two of the compounds and identified them as isochromophilone Ⅵ and sclerotiorin C.6.Increased expression of core genes in dual PKS gene clustersq RT-PCR analyzed the expression levels of three core genes(HR-PKS,NR-PKS,Psaza1)in the WT strain and the OE::PsAza1 mutant strain.Compared with the wild strain,the expression of HR-PKS,NR-PKS and transcription factor Psaza1 was up-regulated by 60-80 times.The innovations of this experiment are:1.Three g1628 deletion mutants were produced through gene knockout,and new peaks of wild strains appeared through HPLC analysis of the fermentation broth of the mutant strains,and the secondary metabolites were separated and purified and finally identified as two types.Indole alkaloids: meleagrin and roquefortine C.As far as we know,this is the first example of obtaining meleagrin and roquefortine C regulating HDAC protein in Penicillium sclerotinum.Our experimental results show that epigenetic modification has great potential in guiding the discovery of biologically active natural products in fungi.2.We conducted gene mining on P.sclerotiorum SD-36 to find a special double PKS gene cluster,and successfully obtained five overexpression mutant strains by overexpression of transcription regulators PsAza1,which were prepared by HPLC separation of the crude substance of the mutant strain’s fermentation broth.Two azaphilones compounds with increased yields compared to wild strains.Azaphilones compounds have a wide range of biological activities and interesting structural features.At the same time,they have good anti-cytotoxicity,anti-viral,anti-microbial,and anti-microbial properties.The anti-inflammatory and anti-cancer activities of these compounds have attracted more and more attention from researchers.更多还原