【作者】 赫荣琳；

【导师】 林福呈；

【作者基本信息】 浙江大学 ， 植物病理学， 2009， 博士

【摘要】 细胞自噬过程是真核生物中普遍存在的非选择性的蛋白降解过程,是真核生物中进化上保守的过程。在细胞的生长和发育过程中,细胞自噬在合成和分解代谢方面起着重要的平衡作用。在特定的条件下,如饥饿条件下,细胞自噬被诱导,胞内的长寿蛋白和细胞器被降解,降解的产物得到再循环利用。这一过程在从酵母到哺乳动物中是非常保守的,其中涉及到的基因的功能在许多真核生物中也非常保守。近来研究发现,在丝状真菌中,细胞自噬过程参与到对细胞外部饥饿条件的应答反应;另外也可能参与到细胞正常的生长和发育过程中。里氏木霉(Trichoderma reesei)是一种重要的工业菌株,在工业生产中主要用于纤维素和半纤维素酶以及其他一些酶类。对里氏木霉的研究主要集中在产酶相关基因上,对细胞自噬过程并未有研究。因此,我们在前人对丝状真菌细胞自噬相关基因的研究基础上,根据同源序列比对,对里氏木霉的细胞自噬相关基因TrATG5进行了克隆,并通过基因敲除的方法对该基因在细胞的生长发育过程中的作用进行了分析。结果如下:1.根据MgATG5预测的氨基酸序列通过同源序列比对,在里氏木霉数据库中发现MgATG5的同源基因,相似度高达59%,定名为TrATG5。2.PCR扩增获得TrATG5基因,并对MgATG5基因的缺失突变体Amgatg5互补,发现MgATG5恢复型突变体的菌落形态、产孢量和致病性都恢复到野生型水平,证明TrATG5与MgATG5基因在功能上是同源的。3.构建TrATG5基因置换载体,通过ATMT转化得到基因TrATG5缺失突变体Atratg5。4.在基因缺失突变体Δtratg5中,观察到液泡内无自噬泡积累,说明细胞自噬过程被阻断;在饥饿条件下,突变体的生长缓慢。5.基因缺失突变体Δtratg5在PDA上气生菌丝明显减少,产孢量显著降;对突变体菌丝和孢子形态的扫描电镜观察发现,与野生型相比,分生孢子梗形态有很大变化,分生孢子形态无显著差异。6.在对纤维素酶产生和蔗糖利用情况测定中,结果显示TrATG5基因的缺失并不影响里氏木霉纤维素酶产生和蔗糖的利用情况。7.构建GFP融合蛋白表达载体,对TrATG5基因的细胞定位进行研究。研究发现TrATG5基因在孢子和菌丝内点状分布。更多还原

【Abstract】 Autophagy is a ubiquitous non-selective degradation process which is conserved in all eukaryotic cells.It is an important pathway to maintain a well-controlled balance between anabolism and catabolism during the cell growth and development. Under some specific circumstances,such as starvation,autophagy is induced.In this process,Long-lived proteins and organelles are degraded and the resulting macromolecule components are reused in the cell development.Autophagy is a highly conserved process among yeast,fungi and mammalians.And genes related to this process are also conserved.Recent studies revealed that autophagy appears to be involved in nutrients recycling during starvation in filamentous fungi,and it has been suggested that it may also be involved in normal developmental processes.Trichoderma reesei is an important industrial strain for the cellulase and hemicellulase production.Mainly researches of T.reesei were focused on genes involved in enzymes production and regulation.The process of autophagy in T.reesei remains uncovered.In the present study,we isolated the TrATG5 gene which is a homolog of MgATG5 gene.The functional analysis of the TrATG5 gene was performed by targeted gene disruption method and the role of this gene in the developmental process was discussed.The homologue in T.reesei was isolated by blastp using the predicted amino acid sequences of MgATG5 in T.reesei database,which designated as TrATG5.The full length of TrATG5 cDNA sequence was obtained by PCR and the complementation vector was constructed for complementing the△mgatg5 mutant.TrATG5 restored the colony character,sporulation and the pathogenicity of the△mgatg5 mutant which indicated that the TrATG5 gene were functional homologue of MgATG5 gene.The△tratg5 mutant was generated by targeted gene disruption method. Autophagic bodies were only observed in the vacuoles of the wild type strain,while it was absent in the vacuoles of the△tratg5 mutant,indicating that autophagy was blocked.The colony of the△tratg5 mutant appeared significant different from the wild type strain due to the defects in sporulation.In the△tratg5 mutant,the conidiophores and the differentiation of the phialides were abnormal,but the morphology of the conidia was not changed.Also the△tratg5 mutant was sensitive to starvation conditions,while the sugrose utilization and the cellulase production of the△tratg5 mutant were not influnced by the depletion of TrATG5 gene.Expression of GFP-ATG5 fusion protein was spot like distributed in the mycelia and the conidia.更多还原