构巢曲霉胞质分裂SIN途径反向调节基因的研究

【作者】 仲国维；

【导师】 陆玲；

【作者基本信息】 南京师范大学 ， 微生物学， 2013， 博士

【摘要】 细胞分裂(cell division)过程是真菌(fungi)进行无性繁殖的重要方式,对于真菌的形态建成是必需的。细胞分裂一旦失调将会导致真菌的生长受损甚至死亡。研究这个过程的发生机制,对于控制医学和农业上有害真菌的增殖或促进工业上有益真菌的生长都具有非常重要的意义。真菌的胞质分裂(cytokinesis)的发生是由母细胞通过收缩肌动蛋白环从而分裂其细胞质,最终产生两个子细胞的过程。胞质分裂是有丝分裂细胞核分裂之后的最后也是最关键的一步。无数的研究表明,该过程需要一个非常保守的信号网络的激活,这个信号网络在芽殖酵母中称为有丝分裂退出网络(Mitotic Exit Network, MEN),在裂殖酵母中称为隔膜形成网络(Septum Initiation Network SIN)。虽然不同的真菌会有不同的机制来完成胞质分裂的过程,但在哺乳动物及构巢曲霉(Aspergillus nidulans)等真菌中的研究已有越来越多证据表明这个网具有一定的保守性。‘与单细胞的酵母不同,丝状真菌构巢曲霉的菌丝是由隔膜所间隔的多核细胞。在构巢曲霉的孢子萌发过程中,分生孢子首先经过多轮的核分裂生成8到16个核,但是直到细胞达到一定的体积后才会产生隔膜,第一个隔膜通常在孢子头和芽管的分隔处生成。因此,菌丝中的胞质分裂和有丝分裂不是同步进行的。在构巢曲霉中SIN途径的SEPH蛋白是由温度敏感型菌株筛选实验发现的,是裂殖酵母SIN信号途径中的丝-苏氨酸激酶Cdc7p的同源蛋白,它是胞质分裂早期所必须的组分。但是目前关于SEPH的调节元件是否存在,以及它们是正向调控还是反向调控SEPH知道的很少。为了对这一机制进行研究,前期实验中我们通过紫外诱变获得了116株能够恢复sepH缺陷的突变菌。通过杂交分离、回交验证、互补实验(complementation test)并测序得到了反向调节基因(suppressor)磷酸核糖焦磷酸合成酶Anprs1(AN6711.4, Phosphoribosyl pyrophosphate, PRPP synthetase)。本课题首先对该基因单独设计引物扩增并连接入质粒Prg3-AMA1-Not I,再次进行互补实验转入菌株Sinl10仍旧得到了和前期相同的结果。这说明Sin110的病态表型确实是由Anprs1基因造成的。进一步通过反向遗传学验证Anprs1基因是sepH的反向调节子,我们构建了乙醇启动子控制的GFP标记的Anprs1同源整合菌株、透射电镜观察、Anprs1完全敲除和C端部分敲除等验证了Anprs1的功能,又通过PRS酶活测定、qRT-PCR技术、酵母双杂交技术验证了Anprs1,2,3家族蛋白的功能。结果显示AnPRS1蛋白弥散定位在胞质中,在正在形成的隔膜上有微弱的聚集信号,推测其在胞质近隔膜的位置参与隔膜形成。抑制表达情况下和Sin110菌株表型一致,电镜结果显示Anprs1基因受到抑制时隔膜呈弯曲状。Anprs1完全敲除菌表型和野生型一致,但是C端敲除菌表型和Sin110菌株一致。AnPRS酶活测定显示Sin110菌株中酶活最低,显示了正常的胞质分裂和PRS酶活相关。但Anprs1,2,3基因测序都没有突变,我们推测酶活的降低可能和这三个基因的转录有关,Sin110菌株的qRT-PCR的结果显示Anprs1,Anprs2和Anprs3都明显表达下调,把这三个基因全部克隆入载体Prg3-AMA1-Not Ⅰ,共转入Sin110菌株,结果其病态表型被完全弥补。进一步的酵母双杂交实验表明构巢曲霉中存在AnPRS家族形成三聚体发挥功能。另外根据酵母中对蛋白磷酸酶2A(protein phosphatases2A, PP2A)调节亚基能够反向调节SIN的发现及其和AnPRS家族都能转移磷酸基团的功能。我们找到并研究了构巢曲霉中PP2A的两个调节亚基parA和pabA,虽然不能反向调节sepH,但对于构巢曲霉的形态建成起重要作用。本课题对于构巢曲霉胞质分裂的研究,将为我们在实际应用中抑制或促进真菌的无性繁殖提供重要的理论基础,对医学上控制肿瘤治疗提供强有力的理论依据。更多还原

【Abstract】 Cell divison is the main way of vegetative propagation for fungi and failure of cell division may be lethal for both, mother and daughter cells. Study on mechanism of this process has a very important significance for the control of medical and agricultural harmful fungi proliferation or promote industry beneficial fungi growth.Cytokinesis is the process by which a cell splits its cytoplasm, accomplished by the contraction of a contractile actin ring, to produce two daughter cells. Accordingly, cytokinesis is the final step in cell division after the nuclear division of mitosis. Numerous studies have identified that mitotic exit requires the activation of the conserved signalling network, termed the mitotic exit network (MEN), in budding yeast and the septation initiation network (SIN) in fission yeast. Although organisms of different kingdoms have developed unique mechanisms to execute cytokinesis, signals that trigger the onset of cytokinesis are evolutionarily conserved. Unlike yeast, the filamentous fungus Aspergillus nidulans contains a mycelium of multinucleate cells that are partitioned by septa. During the germination in A. nidulans, the conidiospores undergo multiple rounds of nuclear division to produce eight or16nuclei in germlings, but they do not undergo septation until the cell reaches an appropriate size/volume, and then forms the first septum near the neck between spore and germ tube. Therefore, as a whole, inter-compartment development and mitosis in the mycelium becomes asynchronous.The serine/threonine protein kinase SEPH in A. nidulans is a Cdc7p orthologue from fission yeast which was first cloned in a screen for temperature-sensitive cytokinesis mutants. It has been identified that SEPH plays a central part in the initiation of septation. However, little is known about how the SEPH kinase cascade is regulated by other components, or whether there exist any of the negative regulators that act antagonistically to others in the SIN. To gain insight into the regulatory mechanisms that underlie septation,116mutants that suppressed the defects of sepH in A. nidulans were isolated by UV mutation in previous study. By cross, backcross, complementation test and sequencing we identified a gene Anprs1(AN6711.4).In this thesis, we first coloned Anprsl using autonomous plasmid replication vector prg3-AMAl-Not I and transformed to strain Sin110. Results showed the same phenotype with previous study. This means phenotype of Sin110was caused by Anprsl. To further confirm how AnPRS1functions, we used a conditional strain in which the Anprsl gene was under the control of the inducible/repressible alcA promoter, TEM, deletion and C-deletion strains creation, and by qRT-PCR, AnPRS activity assay and Y2H to test AnPRS family function during cytokinesis in Aspergillus nidulans. GFP-AnPRS1appeared at the predicted septation site possibly prior to a detectable septum formation. AnPRS1may function in the cytosol and septation sites. When depressed, it displayed a number of phenotypic similarities to that of the Sinl10and transmission electron microscopy showed that, when the repression of AnPRS1showed the aberrant formation of delocalized septa. Anprsl full ORF deletion mutants display normal timing of cytokinesis while a C-terminal truncated displayed the same phenotype to Sinl10. Biochemical assays to test PRPP synthetase activity were performed and results indicated that measurable PRPP synthetase activities in extracts from Sinl10caused a mostly severe defect. Accordingly, normal cytokinesis seems to depend on a normal level of PRPP synthetase activity. While no mutation was found in Anprs genes in Sinl10, we wondered whether the decreased AnPRS activity in Sinl10was related to the transcription of the Anprs family. As expected, by real-time qPCR, three members of the Anprs family in A. nidulans were dramatically downregulated in Sinl10. In response to these results, we cloned ORFs of Anprsl, Anprs2and Anprs3into the AMA1vector to make the plasmids pAMA1-Anprs1, pAMA1-Anprs2and pAMAl-Anprs3respectively. The defects of Sinl10in growth and septation can be dramatically rescued.Lastly, based on the role of PP2A (protein phosphatases2A) negatively regulation SIN in yeast and transferring pi group, we searched its two regulatory subunits, par A and pabA. Though they can’t surppress sepH mutation, they are essential for morphogenesis of Aspergillus nidulans.This study will provide an important theoretic foundation for us in practice promote or inhibit fungal asexual reproduction, the medicine control and provide a strong theoretical basis for treatment of tumor.更多还原