【作者】 胡阳；

【导师】 朱旭东；

【作者基本信息】 南开大学 ， 微生物学， 2013， 博士

【摘要】 球毛壳菌(Chaetomium globosum)是自然界中分布最广泛的真菌之一,因产生球毛壳菌素(chaetoglobosins)而被人们所熟知。球毛壳菌素属于细胞松弛素(cytochalasans)类,能够与细胞内肌动蛋白微丝结合,抑制肿瘤细胞的运动和分裂。然而,球毛壳菌素合成的分子机制,如参与其合成途径的关键基因、影响合成的信号途径均不清楚。球毛壳菌还具有高效分解利用纤维素的能力,但其纤维素酶系统的分子生物学研究基本还是空白。此外,球毛壳菌还可以作为一种植物病害广谱生物防治真菌。本研究首先利用聚酮合酶(polyketide synthase)基因pks-1在C. globosum中成功建立了RNAi的方法,并证实此基因参与球毛壳菌素A (chaetoglobosin A, ChA)的合成。ChA是球毛壳菌众多次生代谢产物中最主要的成分,是由9个单位的乙酰／丙二酰缩合而成的聚酮骨架组成的C18聚酮化合物。RNA干扰pks-1后,ChA的产量明显下降,同时沉默转化子的黑色素合成降低、菌丝变白。表明两种聚酮类化合物—黑色素和球毛壳菌素,可能共有相同合成步骤。本研究还发现pks-1在孢子生成过程中也起着重要的作用,pks-1基因沉默转化子失去了产孢的能力,聚酮类化合物可能通过未知过程调控孢子生成。C. globosum的pks-1基因的多重作用未有报道。本研究为C. globosum的球毛壳菌素生物合成以及次生代谢和真菌形态发生的关系提供有用信息。G protein-cAMP-PKA信号转导通路已经在许多真菌中研究,并且被认为是调节次生代谢的最重要的信号通路之一。本文为研究该信号通路是否控制ChA的合成,通过RNAi方法沉默了编码G蛋白Ⅰ型α亚基的基因gna-1和蛋白激酶A的调节亚基基因pkaR,并获得了gna-1基因沉默转化子(pG)和gnal/pkaR双沉默转化子(pGP)。 gna-1基因沉默转化子出现了与pks-1基因沉默转化子类似的表型,黑色素和孢子合成降低、菌丝颜色变浅,同时,ChA的含量明显下降。gnal/pkaR双沉默转化子则恢复了黑色素和ChA的含量。表明G protein-cAMP-PKA信号转导通路参与C. globosum NK102孢子发生和球毛壳菌素生物合成过程。C. globosum NK102也是一株高效降解纤维素的菌株,本文同时研究了此菌的纤维素酶表达系统并寻找影响酶基因表达的关键因素。通过对NK102测序,本文界定了球毛壳菌NK102的主要纤维素酶编码基因,使用转录组高通量测序RNA-Seq的方法得到纤维素酶基因的表达差异,然后观察了不同营养、物理条件下纤维素酶基因表达和酶活性变化的情况。发现随着培养时间的延长,纤维素酶基因整体上表达量升高。外切葡聚糖酶、纤维二糖脱氢酶和内切葡聚糖酶基因(cbhl,cdh和egl1)的表达量最高。糖代谢的负调控因子ACE1和CreA的表达量均降低,而Hap2/3/5复合体的表达量反而升高。之后检测了不同碳源培养基对纤维素酶基因表达量和酶活性的影响,发现葡萄糖为强抑制因子,纤维二糖为其诱导物,而山梨醇没有影响。特别是,我们发现光照也影响纤维素酶基因的表达,黑暗条件明显抑制纤维素酶基因的表达。转录组学的方法可以初步探索纤维素酶表达的规律,酶基因的表达受到营养、物理条件的影响。本研究为揭示球毛壳菌降解纤维素分子机理和阐释生物质糖代谢途径提供了有用参考。本文还发现G蛋白α亚基GNA1介导的cAMP通路调控NK102中纤维素酶基因的表达。RNA-Seq结果显示gna-1基因沉默转化子的糖代谢过程的基因基本都是下调的。使用qRT-PCR证明几个重要的纤维素酶基因cbhl、cbh2、egll和egl2的表达都下调。gnal/pkaR双沉默转化子或者加入cAMP的类似物能够恢复这些基因的表达量。纤维素培养基表型和酶活性检测等多方面的实验结果充分证实G蛋白-PKA-cAMP通路调控着纤维素碳源代谢,影响纤维素内切酶和外切酶活性。此外,本文首次发现了C. globosum发酵液中具有杀线虫活性的物质为ChA,ChA对南方根结线虫(Meloidogyne incognita)二龄幼虫J2具有强烈的致死效果,ChA的杀线虫能力(LC5o=77.O μg/mL)与合成杀线剂carbofuran基本持平。C.globosum NK102能够显著趋避二龄幼虫。发酵液和ChA对二龄幼虫有致死效果,ChA浓度为300μg/mL时,致死率为90.2%,此时半致死浓度LC50=126.5然而二者对卵孵化的作用直到72h处理后才有显著性影响。所有稀释浓度的发酵液都抑制二龄幼虫的侵染,即使是12.5%浓度的处理也有影响。与对照处理的植物相比,加入浓度为30mg/kg ChA的处理,其卵的数量降低了63%。C. globosumNK102的杀线虫特性,使C. globosum及其代谢物具有开发成杀虫农药的潜力与可能。特别是对南方根结线虫的杀线虫作用,显示出其良好的应用开发前景。更多还原

【Abstract】 Chaetomium globosum is one of most common fungi in nature. It is best known for producing chaetoglobosins, which belong to cytochalasans and inhibit movement and proliferation of mammalian cells due to their ability to bind to actin filaments. However, the molecular basis of chaetoglobosin biosynthesis including the key genes and the signaling pathways is poorly understood’in this fungus. C. globosum also have the ability to decomposite the cellulose effectively, but the molecular basis of cellulase system has remained uninvestigated. In addition, C. globosum has been widely investigated as a method for biological control.In this study, we first use a polyketide synthase gene, pks-1, to establish the technique of RNA interference(RNAi) in C. globosum and then characterized that this gene is involved in the production of chaetoglobosin A(ChA). ChA is the major component of the secondary metabolites in C. globosum and consists of a polyketide backbone that is condensed from nine units of acetate/malonate. When pks-1was knocked down by RNAi, the production of ChA dramatically decreased. Knock-down mutants also displayed a pigment-deficient phenotype. These results suggest that both polyketides, melanin and chaetoglobosin, are likely to share common biosynthetic steps. Most importantly, we found that pks-1also plays a critical role in sporulation. The silenced mutants of pks-1lost the ability to produce spores. We propose that polyketides may modulate cellular development via an unidentified action. Our results suggest that C. globosum pks-1is unique because of its triple role in melanin formation, chaetoglobosin biosynthesis and sporulation. This work implies an interconnection of chaetoglobosin biosynthesis, secondary metabolism and fungal morphogenesis.G protein-cAMP-PKA signaling pathway has been studied in numerous fungi and is recognized as one of the most important signaling pathways regulating secondary metabolite production. To investigate whether this signaling pathway regulate chaetoglobosin biosynthesis, in this study, a G-protein a-subunit-encoding gene, gna-1(G-protein alpha-subunit1), which encodes a protein showing a high degree of identity to Group Ⅰ a-subunits of fungal heterotrimeric G-proteins, was knock down by RNAi. Dozens of gnal-knock down strains (pG) and gnal/pkaR-knock. down strains (pGP) were obtained. The gna-1knock down mutants displayed a pigment-deficient phenotype which was similar to pks-1knock-down mutants. In the meanwhile, the concentration of ChA in the gnal knock-down transformants all fell sharply. These results suggest that melanin production, sporulation and ChA biosynthesis are all regulated by the G protein-cAMP-PKA signaling pathway.C. globosum also have the ability to decomposite the cellulose effectively. This work explored the cellulose-degradation system and factors on cellulase gene expression in Chaetomium globosum NK102. In the sequenced genome of NK102, we identified10cellulase genes by sequence homology alignment. We employed a high-throughput sequencing technology, RNA Sequcing (RNA-Seq), to monitor the differential expression of the genes under different culture conditions. We observed that cellulase activity generally increased with the time of the fungal cultures. Transcription level of the genes encoding cellobiohydrolase, cellobiose dehydrogenase and endoglucanase (cbhl, cdh and egll) was higher than the others. Expression of the transcriptional repressors, ACE Ⅰ and CreA, decreased with the culture age, whereas expression of Hap2/3/5complex was upregulated. In different carbon sources, cellulase activity and their gene transcription were repressed by glucose and were activated by cellobiose. While, sorbitol had no significant effect. Interestingly, light had a positive effect on the expression of these cellulase genes. This study detects the molecular system of cellulose-degradation in C. globosum and provides information for interpretation of carbohydrate metabolism.We also found that G protein-cAMP-PKA signaling pathway regulates cellulase gene expression in C. globosum NK102. RNA-Seq results showed that the expression of most carbon metabolism genes were down-regulated and the qRT-PCR result demonstrated cbh1、cbh2、egl1and egl2expression in gnal knock-down strains (pG) decreased sharply. The gnal/pkaR double knock down strains (pGP) or the activator of the cAMP pathway can restore the cellulose gene expression. In addition, the phenotype of the fungus grown on the cellulose medium and the cellulase activity results confirmed that cellulose degradation are regulated by the Gprotein-cAMP-PKA signaling pathway.The nematicidal activity of C. globosum NK102, culture filtrates and chaetoglobosin A (ChA) purified by HPLC were evaluated on Meloidogyne incognita. The result showed that C. globosum NK102significantly repelled second-stage juveniles (J2). Both filtrates and ChA demonstrated strong adverse effects on J2mortality of90.2%at300μg ChA/mL (LC50=126.5μg/mL). ChA and filtrates did not affect egg hatch until72h exposure. All filtrate treatments inhibited the J2penetration even in12.5%dilution treatment. Similarly, ChA (300and30μg/mL) showed a significant inhibitory effect on J2penetration. The number of egg per plant was significantly reduced by63%in the treatment of30mg ChA per kg soil relative to control plants, indicating an apparent negative effect on reproduction of M. incognita. The report suggests a potential for C. globosum as biocontrol agent for the integrated management of M. incognita.更多还原