少孢节丛孢中5个生物合成基因与真菌形态和次生代谢产物组学关联的初步探究

发布时间：2018-03-08 14:53

本文选题：少孢节丛孢　切入点：生物合成基因　出处：《云南大学》2016年硕士论文　论文类型：学位论文

【摘要】：本文以捕食线虫真菌的模式菌株少孢节丛孢(Arthrobotrys oligospora)中的1个Ⅰ型聚酮合酶(PKS)基因AOL_s00079g496、1个萜类合成酶(TPS)基因AOL_s00079g224及3个细胞色素P450基因AOL_s00079g225、AOL_s00043g740和AOL_s00210g57为研究对象,通过敲除质粒构建和原生质体转化方法获得了6个敲除突变菌株。通过对这6株突变菌株与野生菌株在菌丝形态、菌丝生长直径、产孢量、孢子萌发、三维菌网生成和捕食线虫能力及其次生代谢产物图谱等方面比较分析,初步探究了这些生物合成基因对少孢节丛孢次生代谢产物图谱及其形态、侵染能力的影响。表型比较结果显示,这6株突变株与野生菌株均有不同程度的差异,其中PKS基因AOL_s00079g496-KS和两个P450基因AOL_s00079g225和AOL_s00210g57的突变株表型差异较为突出,在菌丝形态上,与野生菌株相比这三株突变菌的菌丝明显茂盛,其中突变株MOL_s00079g496-KS最为显著,其它菌株则无明显差异。在菌丝生长直径上,与野生菌株相比PKS基因突变株△AOL_s00079g496-KS及 △AOL_s00079g496-KR在富营养培养基TYGA平板上生长稍慢,另一株TPS基因突变株△AOL_s00079g224在乏营养培养基CMA平板上菌丝生长直径也明显小于野生菌株,P450基因突变株△AOL_s00079g225、AAOL_s00210g57及△AOL_s00043g740则无显著差异。在产孢量上,除△AOL_s00210g57的产孢量与野生菌株无明显差异外,其他突变株产孢量均高于野生菌株,增加范围为58%-300%,其中突变株△AOL_s00079g224最为显著。在孢子萌发上,所有突变株相比野生菌株都有不同程度的减缓,2h时表现最为明显,不同突变菌株的孢子萌发率的减少幅度为24%-70%,其中TPS基因突变株△AOL_s00079g224最为显著。在捕器数量上,P450基因突变株△AOL_s00079g225与野生菌株的捕器数量差异最为显著,在12 h、24 h、36 h分别增加了约49%、35%、20%,其他突变菌株则无显著性差异。在捕食线虫能力上,与野生菌株相比有显著差异的有PKS基因突变株△AOL_s00079g496-KS、 △AOL_s00079g496-KR及P450基因突变株△AOL_s00079g225、△AOL_s002I0g57,其中突变株△AOL_s00079g496-KS、△AOL_s00079g496-KR在24 h时线虫捕食率明显低于野生菌株,分别降低了约41%和32%；而突变株△AOL_s00079g225与△AOL_s00210g57在12h时线虫捕食率比野生菌株提高了约2倍和3倍,其他突变株则无显著性差异。HPLC检测图谱比较结果显示,Ⅰ型PKS基因AOL_s0079g496的KS结构域敲除后对少孢节丛孢次生代谢产物的影响变化最大,突变株△AOL_s00079g496-KS与野生菌株相比至少缺失了5个峰,新增了8个峰,并且至少有4个峰面积发生了变化；3个P450基因的突变株与野生菌株相比变化的主要是峰面积,其中△AOL_s00079g225峰面积发生变化的峰有7个,△AOL_s00210g57及△AOL_s00043g740峰面积发生变化的峰有11个。GC-MS检测结果显示,AOL_s00079g225敲除后有6个萜类化合物发生变化,因此推测P450基因AOL_s00079g225的功能可能是对TPS基因AOL_s00079g224的产物进行修饰；AOL_s00043g740敲除后,有9个脂肪烷类和5个直链萜类化合物发生了变化,而P450单加氧酶会参与不饱和脂肪酸的代谢及萜类的合成,因此推测P450基因AOL_s00043g740可能对不饱和脂肪酸及直链萜类化合物进行修饰；在PKS基因AOL_s00079g496、TPS基因AOL_s00079g224及P450基因AOL_s000210g57的突变株中没有检测到哪一类化合物明显发生变化,因此它们具体参与哪些化合物的合成还有待进一步研究。值得注意的是,发现在三株P450基因的突变株发酵液中都检测到了5-methyl-2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)be-nzene-1,3-diol这个化合物,它与少孢节丛孢中已经鉴定的节丛孢素类化合物有相似的骨架结构,因此推测这三个P450基因参与的合成途径可能与这类化合物相关。本论文研究发现,在五个三种不同类型的生物合成基因中,对形态和捕食能力影响较大的基因的有Ⅰ型PKS基因AOL_s00079g496和P450基因AOL_s00079g225、 AOL_s00210g57,对次生代谢产物影响较大的为Ⅰ型PKS基因AOL_s00079g496和TPS基因AOL_s0079g224。其中PKS基因AOL_s00079g496敲除后少孢节丛孢在形态和次生代谢产物上都有较大变化,P450基因AOL_s00079g225与AOL_s00210g57敲除后对少孢节丛孢的影响则主要表现在形态上。以上结果表明,少孢节丛孢中部分生物合成基因与其形态及次生代谢组学相关联,本论文也是首次发现捕食线虫真菌中的Ⅰ型PKS基因及P450基因能参与调控其菌丝形态、捕器形成及捕食线虫能力。
[Abstract]:Based on the model strain of nematode trapping fungus a.oligosporus (Arthrobotrys oligospora) 1 type of polyketide synthase (PKS) gene AOL_s00079g496,1 terpene synthase (TPS) gene AOL_s00079g224 and 3 cytochrome P450 genes AOL_s00079g225, AOL_s00043g740 and AOL_s00210g57 as the research object, through the knockout plasmid and protoplast conversion method to take 6 knockout mutant strains. Based on these 6 mutant strains and wild strains in mycelial morphology, mycelial growth diameter, sporulation, spore germination, comparative analysis of 3D mesh generation and predator nematode bacteria ability and secondary metabolites of atlas, preliminary explore these biosynthetic genes for less Arthrobotrys secondary metabolites map and its morphology, affect the infection ability. The phenotypic comparison shows that the 6 mutant strains and wild strains had difference in different degrees, the In the PKS AOL_s00079g496-KS gene and two P450 genes AOL_s00079g225 and AOL_s00210g57 mutant phenotypes is more prominent in mycelial morphology, compared with wild strains of these three mutant strains of mycelium was lush, including the mutant MOL_s00079g496-KS was the most significant, and there were no significant differences in other strains. The diameter of mycelial growth, compared with wild strain the PKS mutant Delta AOL_s00079g496-KS and delta AOL_s00079g496-KR medium plate TYGA growth slightly slower in rich nutrition, another strain of TPS mutant AOL_s00079g224 CMA medium plate on the mycelial growth of smaller diameter than the wild strain in the lack of nutrition, the P450 mutant AOL_s00079g225, there is no significant difference between AAOL_s00210g57 and AOL_s00043g740. In sporulation, in addition to no significant difference between the sporulation of delta AOL_s00210g57 and wild strain and other mutant sporulation were Higher than that of the wild strain, increase the range of 58%-300%, the mutant AOL_s00079g224 was the most significant. In spore germination, all mutant wild strains had slowed to varying degrees, 2h was the most obvious, the germination rate of spores of different mutant strains reduced the amplitude of 24%-70%, the TPS gene mutant Delta AOL_s00079g224 significant. In trap number, P450 mutant AOL_s00079g225 and wild strain of the trap number difference was most significant at 12 h, 24 h, 36 h were increased by about 49%, 35%, 20%, other strains had no significant difference. The nematode trapping ability, compared with the wild there is a significant difference of strain PKS mutant Delta AOL_s00079g496-KS, Delta AOL_s00079g496-KR and P450 mutant Delta AOL_s00079g225, Delta AOL_s002I0g57, the mutant Delta AOL_s00079g496-KS, Delta AOL_s00079g496-KR at 24 h Nematode predation rate was significantly lower than that of the wild strain, were reduced by 41% and 32%; and delta AOL_s00079g225 and delta AOL_s00210g57 mutant in 12h nematode predation rate than wild strain increased by about 2 times and 3 times the other mutants had no significant difference.HPLC detection map comparison results show that the KS domain of PKS the biggest change in AOL_s0079g496 gene knock on effect on a.oligosporus secondary metabolites of mutant AOL_s00079g496-KS, compared with the wild strain missing at least 5 peaks, 8 new peaks, and at least 4 were changed to a peak area; 3 P450 gene mutant compared with the wild strain changes is the main peak area of the delta AOL_s00079g225 peak area changes the peak of 7, Delta AOL_s00210g57 and delta AOL_s00043g740 peak area changes the peak 11.GC-MS test results showed that AOL_s00079g225 knockout There are 6 terpenoids change, suggesting that P450 gene AOL_s00079g225 may function is the product of TPS gene modified AOL_s00079g224; AOL_s00043g740 knockout, has changed 9 fat 5 straight chain alkanes and terpenes, and P450 monooxygenase in unsaturated metabolism and synthesis terpene fatty acids, suggesting that P450 gene AOL_s00043g740 may be of unsaturated fatty acid and straight chain terpenoids were modified; in the PKS AOL_s00079g496 gene, TPS gene mutation and P450 gene of AOL_s00079g224 AOL_s000210g57 were not detected in which compounds have changed obviously, so they are specifically involved in the synthesis of compounds which have yet to be further research. It is worth noting that the mutant found in the fermentation liquid of P450 genes of three strains were detected by 5-methyl-2- ((2E, 6E) -3,7,11-trimethyldodeca-2 6,10-trien-1-yl, be-nzene-1,3-diol) of this compound, it has Baosu Arthrobotrys compounds and identification of Arthrobotrys oligospora Baozhong has the skeleton structure similar, suggesting that the synthesis pathway of three P450 genes may participate with these compounds. This study found that in five of three different types of biosynthetic genes. The morphology and predation ability influence gene with type I PKS gene AOL_s00079g496 and P450 gene AOL_s00079g225, AOL_s00210g57, on the secondary metabolites of type I PKS gene AOL_s00079g496 and TPS gene AOL_s0079g224. PKS AOL_s00079g496 gene knockout a.oligosporus have great changes in morphology and secondary metabolites, P450 AOL_s00079g225 and AOL_s00210g57 gene knock of a.oligosporus after is mainly manifested in the form. The above results show that less spore Festival Learn the associated part of Baozhong plexus biosynthesis gene and its morphology and secondary metabolism group, this thesis is the first type of PKS gene and P450 gene in nematode trapping fungi can participate in the regulation of the mycelial morphology, trap formation and nematode trapping ability.

【学位授予单位】：云南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476

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